Ligands for nematode nuclear receptors and uses thereof

ABSTRACT

Anti-nematode compounds, compositions, and methods for identifying such compounds are disclosed, where the compounds have the formula I: 
                         
where Q, Q′, R 1 , R 2 , and n are defined herein.

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 60/780,050, filed on Mar. 8, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of anti-nematodecompounds and methods of using the same.

2. State of the Art

Nematodes are elongated symmetrical roundworms that constitute one ofthe largest and most successful phyla in the animal kingdom. Manynematode species are free-living and feed on bacteria, whereas othershave evolved into parasites of plants and animals, including humans.Human infections with parasitic nematodes are among the most prevalentinfections worldwide. Over one billion people, predominantly in tropicaland subtropical developing countries, are infected with soil andvector-borne nematodes that cause a variety of debilitating diseases.Liu et al., “Intestinal Nematodes” in 181 HARRISON'S PRINCIPLES OFINTERNAL MEDICINE 916-20 (McGraw-Hill, 1994).

Among these parasitic nematodes are Ancylostoma and Necator hookworms,which cause anemia and malnutrition, Ascaris roundworms, that can causepulmonary and nutritional disorders, and Strongyloides stercoralis, thatcan effect a potentially life-threatening intestinal infection.Nematodes of the order Spirurida are responsible for onchocerciasis(river blindness) and lymphatic filariasis. Animal parasitic nematodesinfect a wide variety of both domestic and wild animals. Major animalpathogens include Haemonchus contortus, which infects herbivorousvertebrates, Trichinella spiralis, the causative agent of trichinosis,and various members of the order Ascaridida, which infect pigs and dogsin addition to humans.

Plant parasitic nematodes also represent major agricultural problems andare responsible for many billions of dollars in economic lossesannually. The most economically damaging plant parasitic nematode generabelong to the family Heterderidae of the order Tylenchida, and includethe cyst nematodes (genera Heterodera and Globodera) and the root-knotnematodes (genus Meloidogyne). The soybean cyst nematode (H. glycines)and potato cyst nematodes (G. pallida and G. rostochiensis) areimportant examples. Root-knot nematodes infect thousands of differentplant species including vegetables, fruits, and row crops. In contrastto many viral and bacterial pathogens, little is known about themolecular basis of nematode parasitism, limiting the available frameworkfor rational anti-helminthic (anti-nematode) drug development. See Davidand Liu, “Molecular biology and immunology of parasitic infections,” inHARRISON'S PRINCIPLES OF INTERNAL MEDICINE 865-71 (McGraw-Hill, 1994).

Anti-nematode drug or pesticide discovery traditionally has relied ondirect screening of compounds against whole target organisms or onchemical modification of existing compounds. These strategies haveyielded relatively few classes of agents, acting against a limitednumber of known biological targets. For example, organophosphates andcarbamates, the oldest extant class of nematicides, were developed manydecades ago and target a single, biologically conserved enzyme,acetylcholinesterase. Imidazole derivatives such as benzimidazole exerttheir antiparasitic effects by binding tubulin. Levamisole acts as anagonist on the nicotinic acetylcholine receptor, and avermectins act asirreversible agonists at glutamate-gated chloride channels (Liu et al.,1996).

Unfortunately, there are certain debilitating nematode infections thatare difficult if not impossible to cure with existing therapeutics. Inonchocerciasis, for instance, the adult female Onchocerca volvulus wormsare refractory to even newer generation drugs (Liu et al., 1996). Inaddition, drug resistance has emerged to all of these main classes oftherapeutics, particularly in livestock animal applications in whichtheir use is widespread (Sangster et al., 1999). To date it has not beenpossible to develop effective and practical vaccines. Even were suchvaccines available, effective anti-nematode drugs still would be needed,for treating established infections and for offering the potentialadvantages of prophylaxis and treatment against a broad spectrum ofnematode parasites.

The drawbacks of existing agents that are currently used to controlplant parasitic nematodes are equally or more significant. Fumigantnematicides such as methyl bromide and 1,3-dichloropropene, which killnematodes by slowly diffusing through the soil, are phytotoxic and mustbe applied well before planting. Environmental concerns, primarilygroundwater contamination, ozone depletion, and pesticide residues infood (National Research Council, Pesticides in the Diet of Infants andChildren (Washington, D.C.: National Academy of Sciences, 1993) haveprompted the removal of Aldicarb, DGBCP, and other toxic nematicidesfrom the market by the Environmental Protection Agency, with methylbromide to be withdrawn in the U.S. by 2002. Johnson & Bailey,“Pesticide Risk Management and the United States Food Quality ProtectionAct of 1996,” in PESTICIDE CHEMISTRY AND BIOSCIENCE: THEFOOD-ENVIRONMENT CHALLENGE 411-20 (Royal Society of Chemistry,Cambridge, 1999). Physical control measures, such as solarization andhot water treatment, crop rotation and other biological controlmeasures, and integrated approaches have been used to ameliorate thedamage caused by plant parasitic nematodes. See, e.g., Whitehead, PlantNematode Control, Wallingford: CAB International (1998). No singlemethod or combination of measures is uniformly effective, however.

Molecular genetic methods, such as gene knockouts, can uncover thebiological function of individual genes and proteins in an organism,information that can form the foundation for developing target-basedcompound discovery screens. At present, however, these techniques aredifficult to perform in parasitic nematodes.

In contrast, such procedures can be performed in a straightforwardmanner in C. elegans. Furthermore, the complicated life cycle of manyparasitic nematodes and their need for a suitable plant or animal hostmakes it inconvenient to propagate them in the laboratory.

The genome of C. elegans is predicted to contain 284 nuclear receptors(Gissendanner et al., 2004; Sluder and Maina, 2001). Forward and reversegenetic studies have uncovered roles for C. elegans receptors in diversephysiological processes, such as dauer formation, reproduction, and lifespan (DAF-12), larval molting (NHR-23), sex determination (SEX-1),xenobiotic metabolism (NHR-8), neuronal development (UNC-55, ODR-7,FAX-1) and lipid metabolism (NHR-49). Nevertheless, all nuclearreceptors in worms remain “orphans,” since ligands regulating theirfunction have not been identified (Lindblom et al., 2001; Sluder andMaina, 2001; Van Gilst et al., 2005a; Van Gilst et al., 2005b).

In contrast to other C. elegans nuclear receptors, a considerable amountof genetic evidence supports the existence of a steroid-like ligand forthe orphan receptor, DAF-12. DAF-12 belongs to a group of over 30 genes,collectively called daf (dauer formation) genes, which transduceenvironmental signals that influence the choice between alternativedevelopmental programs of dauer diapause or reproductive development(Antebi et al., 2000; Riddle and Albert, 1997).

Dauer diapause is a process in which animals at the second larval stage(L2) delay further reproductive development under conditions ofdiminishing food or overcrowding and instead form the non-feeding,non-reproductive, and long-lived dauer larva (Riddle and Albert, 1997).Upon entry into a more favorable environment, dauer larvae resumefeeding and reproductive growth.

Mutations in Daf genes generally produce a dauer constitutive phenotype(Daf-c) or a dauer defective phenotype (Daf-d). Daf-c mutants alwaysarrest as dauers, while Daf-d mutants bypass dauer, regardless ofenvironmental signals. Loss of daf-12 results in Daf-d as well as L3stage heterochronic phenotypes, indicating that daf-12 is required fordauer formation and for proper developmental timing in the reproductivestate (Antebi et al., 1998; Antebi et al., 2000).

Detailed analysis of the dauer formation genes has revealed thatfavorable environments activate insulin/IGF-1 and TGFβ signalingpathways within the organism that converge on DAF-12 to inhibit itsdauer promoting function and activate its reproductive function (Kimuraet al., 1997; Ren et al., 1996; Schackwitz et al., 1996). Acting cellnon-autonomously, these pathways are believed to activate, eitherdirectly or indirectly, the production of a DAF-12 ligand by thecytochrome P450, DAF-9 (Gerisch et al., 2001; Jia et al., 2002).Evidence for this model stems from the findings that insulin-likereceptor (daf-2), TGFβ (daf-7), and cytochrome P450 (daf-9) signalingmutants are Daf-c. Furthermore, epistasis experiments have revealed thatthey act upstream of daf-12, since Daf-d alleles of daf-12 suppress theDaf-c phenotypes exhibited by these signaling mutants (Larsen et al.,1995). In addition to the Daf-d alleles, Daf-c mutants of daf-12 havebeen isolated that map to a single residue (R564) in the putative ligandbinding domain of DAF-12 and are predicted to perturb ligand binding(Antebi et al., 2000). Phenotypically, these mutants arrest as partialdauers but recover and resemble weak daf-9 alleles that exhibit gonadalmigration (Mig) defects (Gerisch et al., 2001; Jia et al., 2002). Thus,the predicted loss of hormone production in daf-9 null worms or loss ofhormone binding by daf-12 Daf-c worms results in a failure to inhibitdauer-promoting functions and activate L3 stage reproductive functionsof DAF-12.

Several lines of evidence suggest that DAF-12 ligands may be derivedfrom cholesterol. First, C. elegans lacks the ability to synthesizecholesterol, which is required exogenously for normal growth andfertility (Chitwood, 1999). Second, cholesterol deprivation produces Migand Daf-c phenotypes in wild-type worms and enhances the Mig and Daf-cphenotypes of weak daf-9 and daf-12 alleles (Gerisch et al., 2001; Jiaet al., 2002; Matyash et al., 2004). Finally, worms lacking bothhomologs (ncr-1, ncr-2) of the human Niemann-Pick type C1 gene, amembrane glycoprotein implicated in lysosomal transport of cholesterol,arrest constitutively as dauers (Li et al., 2004).

These data indicate that a sterol-derived hormone promotes reproductivedevelopment in C. elegans. Evidence that lipid extracts from wild-typeworms can rescue daf-9 phenotypes has strengthened the hormonehypothesis (Gill et al., 2004). Nevertheless, the identities ofDAF-9-derived hormonal ligands that activate DAF-12 have remainedelusive.

Accordingly, ligands must be identified that modulate the DAF-9/DAF-12pathway, thereby to identify new agents that are active againstpathogenic and parasitic nematode species, e.g., compounds activeagainst animal or plant parasitic nematodes. A need also exists for newmethodologies and screening technologies that allow for theidentification of compounds active against nematodes. In particular,screening assays are needed that can be performed conveniently, in ahigh throughput format.

SUMMARY OF THE INVENTION

The present invention provides a novel approach to controlling nematodegrowth and infestation, by influencing the biochemical pathways thatdetermine whether nematodes enter, avoid, or recover from an infectivelarvae stage, illustrated by the dauer stage in C. elegans. Inparticular, antagonist and agonist compounds of certain nuclearreceptors in the pathways are contemplated. On the one hand, antagonistcompounds can induce dauer formation or, for a parasitic nematode,induce the infective stage, thereby to delay or prevent nematodedevelopment and reproduction, pursuant to the invention. On the otherhand, agonists can effect avoidance or recovery of the dauer stage,which, according to the invention, can serve to undercut nematodesurvival, e.g., when an inadequate food supply or other environmentalstress otherwise favors a larval state.

An important aspect of the present invention is the inventors' findingthat the cytochrome P450 enzyme, DAF-9, metabolizes 3-keto steroids into3-keto steroidal acids, which then bind the intracellular nuclearreceptor, DAF-12, to disfavor entry into the dauer stage or allowrecovery from the dauer stage. By the same token, the present inventioncontemplates related synthetic compounds, compositions, and methodologyfor controlling nematode growth and treating nematode infestation. Theinvention further encompasses procedures and assays for identifyingcompounds that modulate the activity of DAF-9 and DAF-12, along withtheir homologous receptors in parasitic nematodes.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures relate the following abbreviation: chenodeoxycholic acid(CDCA), cholic acid (CA), deoxycholic (DCA) acid, lithocholic acid(LCA), 3-keto-lithocholic acid (3K-LCA), 6-keto-lithocholic acid(6K-LCA), 7-keto-lithocholic acid (7K-LCA). Reporter gene activity isexpressed as fold induction of relative light units (RLU) compared toethanol control (n=3±SD).

FIG. 1 (A) shows structures of DAF-12 ligand precursors relative tocholesterol and 3-keto-lithocholic acid.

FIGS. 1 (B and C) shows activation of DAF-12 by 10M bile acids (B) or C.elegans sterols (C) and their 3-keto derivatives in the presence ofDAF-9 (black bars) or in its absence (white bars). In (B)co-transfection of the intestinal bile acid transporter (IBAT)expression plasmid was used to facilitate bile acid uptake.

FIG. 1 (D) shows rescue of daf-9(dh6) null worms by sterols afterincubation with DAF-9 microsomes. Results are reported as percentage ofanimals rescued from dauer as wildtype gravid adults (WT), Mig adults,or molt-defective larvae. Numbers in each bar refer to worms tested.

FIG. 1 (E) shows dose response of DAF-12 activation to indicated sterolsin cells co-transfected with DAF-9.

FIG. 2 (A) shows representative UV chromatogram of 4-cholesten-3-one and(B) shows reconstructed total-ion-current chromatogram of lathosteroneafter incubation of 100 μM substrate with DAF-9 (upper line) or control(lower line) microsomes. Product peaks unique to DAF-9 and theirretention times are indicated by the arrows. IS, internal standard of1,4-cholestadien-3-one.

FIGS. 2 (C-F) HPLC fractions from 10 pooled DAF-9 microsomes incubatedwith 4-cholesten-3-one or lathosterone were tested for GAL4-DAF-12activity in the absence of DAF-9 (C and D), or for rescue of daf-9 nullphenotypes (E and F). Fractions correspond to 1 min intervals ofretention times in (A and B). Transfections and rescue assays aredescribed in FIG. 1 legend. Average number of worms tested in (E) and(F) were 75 and 125, respectively.

FIGS. 2 (G and H) shows mass spectra of DAF-9 metabolites of4-cholesten-3-one (peaks 1-2) and lathosterone (peaks 3-4) scanned fromm/z 250-500.

FIG. 3 (A) shows side chain substitutions of 5-cholesten-3β-ol (Ring Aor delta-5) and 4-cholesten-3-one (Ring B or delta-4) derivatives.

FIG. 3 (B) shows DAF-9 independent activation of GAL4-DAF-12 in HEK293cells after incubation with the indicated sterols (10 μM for all sterolsexcept 22(R)-hydroxy-4-cholesten-3-one, which was 4 μM).

FIG. 3 (C) shows UV chromatogram of 4-cholesten-3-one oxysterols (toppanel) compared to DAF-9 (upper line) or control (lower line) microsomesincubated with 100 μM 4-cholesten-3-one (bottom panel). Arrows indicateco-eluting sterols. The carboxylic acid and alcohol metabolites of DAF-9are indicated.

FIG. 3 (D) shows UV chromatogram of DAF-9 (upper line) and control(lower line) microsomes after incubation with 100 μM(25R),26-hydroxy-4-cholesten-3-one (top panel) or(25S),26-hydroxy-4-cholesten-3-one (bottom panel). Arrows indicateproducts unique to DAF-9 microsomes. Mass spectra of DAF-9 metabolites(insets) were obtained in positive ion scan mode.

FIG. 3 (E) Microsome reactions from (D) were diluted 8-fold and testedfor daf-9 rescue as in FIG. 1D. Numbers indicate worms included in eachexperiment.

FIG. 4 (A) shows structures of 4-cholesten-3-one metabolites of DAF-9

FIG. 4 (B) shows dose response of GAL4-DAF-12 activation to4-cholesten-3-one metabolites in HEK293 cells.

FIG. 4 (C) shows DIC microscopy of daf-9(dh6) (a-f) and daf-9(rh50)(g-h) mutants treated with or without 250 nM(25S),26-3-keto-4-cholestenoic acid. (a) Rescued adult, (b) partialdauer, (c) head of rescued L3 larva, (d) head of partial dauer, (e)cuticle of rescued L3 larva, (f) dauer alae, (g) reflexed gonad of L3larva, (h) unreflexed gonad of L3 larva.

FIG. 4 (D) shows response of daf-9(dh6) nulls treated with(25S),26-3-keto-4-cholestenoic acid or (25R),26-3-keto-4-cholestenoicacid. Results are expressed as percentage of worms rescued from dauerafter 3 days at 20° C. Worms were scored as adults or molt-defectivelarvae.

FIG. 4 (E) shows rescue of daf-9(rh50), daf-12(rh61), and daf-12(rh273)Mig phenotypes by (25S),26-3-keto-4-cholestenoic acid. Results areexpressed as percentage of reflexed gonadal arms scored after 3 days at20° C. (n>60 from 2 experiments).

FIG. 4 (F) shows rescue of daf-9(dh6), daf-2(e)368), daf-7(m62),ncr-1(nr2023)ncr-2(nr2022), daf-12(rh273), and daf-2(e1370) mutants by(25S),26-3-keto-4-cholestenoic acid. Also shown by different shading arethe percentage of dauer-rescued worms that exhibited wild-type adult(black bar) or Mig (striped bar) gonads, or an arrested L3 phenotype(white bar). Dauer rescue was scored after 2 days at 25° C. for daf-2and daf-7, or 3 days at 20° C. for daf-9, daf-12 and ncr-1; ncr-2 (n>200from 2 independent experiments).

FIG. 5 (A) shows ligand-dependent interaction of DAF12 with DIN-1S bymammalian two-hybrid analysis in HEK293 cells co-transfected withGAL4-DIN-1S and VP16-DAF-12 or VP16-DAF-12 R564C.

FIG. 5 (B) shows effect of DIN-1S on basal activation of GAL4-DAF-12 inHEK293 cells with or without 100 nM (25S),26-3-keto-4-cholestenoic acid.Cells were transfected with 45 ng/well DIN-1S and 15 ng/wellGAL4-DAF-12.

FIG. 5 (C) shows mammalian two-hybrid assay in HEK293 cellsco-transfected with GAL4-SRC-1-interaction domain 4 (ID4) andVP16-DAF-12 or VP16-DAF-12 R564C.

FIG. 5 (D) shows dose responsive activation of a luciferase reportercontaining the Lit-1 kinase genomic regulatory region by full-lengthDAF-12 and (25S),26-3-keto-4-cholestenoic acid in HEK293 cells.

FIG. 6 (A-C) shows alpha screen assay for ligand-dependent co-activatorrecruitment to the DAF-12 ligand binding domain, where empty vector(CMX) or CMX VP16 were used as controls. Reactions were performed in thepresence of the indicated sterols (1 μM) (A), with increasingconcentrations of (25S),26-3-keto-4-cholestenoic acid (B), or with a1:5000 dilution of DAF-9 or control microsomes incubated with 100 μMlathosterone. Results are expressed as arbitrary binding units fromtriplicate assays (±SD).

FIG. 6 (D) shows structures of DAF-12 ligands.

FIG. 7 (A) shows a strategy for purifying endogenous DAF-12 agonistsfrom C. elegans lipid extracts.

FIG. 7 (B) shows GAL4-DAF-12 activation in HEK293 cells in the presenceof fractionated lipid extracts.

FIG. 7 (C) shows GAL4-DAF-12 activation in HEK293 cells by silicafractions of lipids eluted with acetone:methanol.

FIG. 7 (D) shows LC/MS analysis of pooled and re-purified fractions57-64 in negative selective ion monitoring mode (m/z 413) compared withDAF-9 metabolites of 4-cholesten-3-one.

FIG. 8. HEK293 cells were co-transfected with GAL4-DAF-12,tk-MH100x-4-Luc, bovine adrenodoxin, and the indicated P450 expressionplasmids or empty expression vector (solid line). 4-Cholesten-3-one andlathosterone were added at 25 μM and activation was compared to vehiclecontrol (n=3±SD).

FIG. 9 shows UV chromatogram and mass spectra of 3-keto-4-cholestenoicacid.

Standards for (25R),26-3-keto-4-cholestenoic acid (upper panel) and(25S),26-3-keto-4-cholestenoic acid (lower panel) are shown. Insets showmass spectra obtained in both positive (left) and negative (right) ionmode.

FIG. 10. Expression plasmids for GAL4-DAF-12, GAL4-DAF-12-R564C,GAL4-DAF-12-R564H, and a panel of vertebrate and invertebrateGAL4-nuclear receptors were tested for activation by 5 μM(25S),26-3-keto-4-cholestenoic acid in HEK293 cells. Results for eachcondition were obtained from triplicate assay (±SD) and are expressed asfold induction relative to ethanol vehicle. RLU, relative light units.

FIGS. 11A and 11B. Chemical structures, names, and ¹H-NMR (400 MHz) datafor compounds disclosed herein.

FIG. 12. Transactivation of C. elegans nuclear hormone receptor DAF-12by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic S acid),3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic R acid), and3β-ol-5-cholesten-25(S)-26-oic acid.

FIG. 13. Transactivation of human hookworm N. americanus nuclear hormonereceptor DAF-12 by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic Sacid), 3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic Racid), and 3β-ol-5-cholesten-25(S)-26-oic acid.

FIG. 14. Transactivation of human/hamster hookworm A. ceyelanicumnuclear hormone receptor DAF-12 by (25S),26-3-keto-4-cholestenoic acid(Δ⁴ dafachronic S acid), 3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷dafachronic R acid), and 3β-ol-5-cholesten-25(S)-26-oic acid.

FIG. 15. Transactivation of dog hookworm A. caninum nuclear hormonereceptor DAF-12 by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic Sacid), 3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic Racid), and 3β-ol-5-cholesten-25(S)-26-oic acid.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments ofthe inventive compounds, compositions, and methodology. The variousembodiments described are representative examples and should not beconstrued as descriptions of alternative species. Rather, thedescriptions provided here may be of overlapping scope. The embodimentsdiscussed are illustrative only and are not meant to limit the scope ofthe present invention.

DEFINITIONS

Unless indicated otherwise, the terms and phrases used in thisdescription have the following meanings:

“Alkyl” refers to a straight or branched chain, saturated hydrocarbonhaving the indicated number of carbon atoms. For example, (C₁-C₆)alkylis meant to include, but is not limited to methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,hexyl, isohexyl, and neohexyl. An alkyl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinthroughout.

“Alkenyl” denotes a straight or branched chain unsaturated hydrocarbonhaving the indicated number of carbon atoms and at least one doublebond. Examples of a (C₂-C₈)alkenyl group include, but are not limitedto, ethylene, propylene, 1-butylene, 2-butylene, isobutylene,sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene, 2-hexene,3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene, isoheptene,1-octene, 2-octene, 3-octene, 4-octene, and isooctene. An alkenyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein throughout.

“Alkynyl” refers to a straight or branched chain unsaturated hydrocarbonhaving the indicated number of carbon atoms and at least one triplebond. Examples of a (C₂-C₈)alkynyl group include, but are not limitedto, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne,1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne,2-octyne, 3-octyne and 4-octyne. An alkynyl group can be unsubstitutedor optionally substituted with one or more substituents as describedherein throughout.

The term “aryl” refers to a 6- to 14-membered monocyclic, bicyclic ortricyclic aromatic hydrocarbon ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinthroughout.

“Cycloalkyl” denotes a 3- to 14-membered saturated or unsaturatednon-aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring system.Included in this class are cycloalkyl groups which are fused to abenzene ring. Representative cycloalkyl groups include but are notlimited to cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, and tetrahydroheptalene. A cycloalkyl group can beunsubstituted or optionally substituted with one or more substituents asdescribed herein throughout.

“Halo” denotes —F, —Cl, —Br or —I.

“Haloalkyl” refers to a C₁-C₆ alkyl group wherein from one or more ofthe C₁-C₆ alkyl group's hydrogen atom is replaced with a halogen atom,which can be the same or different. Examples of haloalkyl groupsinclude, but are not limited to, trifluoromethyl, 2,2,2-trifluoroethyl,4-chlorobutyl, 3-bromopropyl, pentachloroethyl, and1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroaryl” refers to an aromatic heterocycle ring of 5 to 14members and having at least one heteroatom selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, includingmonocyclic, bicyclic, and tricyclic ring systems. Representativeheteroaryls are triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl,benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl,quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyl and oxazolyl.A heteroaryl group can be unsubstituted or optionally substituted withone or more substituents as described throughout.

“Heteroatom” is inclusive of oxygen (O), nitrogen (N), and sulfur (S).

The term “heterocycle” refers to 3- to 14-membered ring systems that areeither saturated, unsaturated, or aromatic, and that contains from 1 to4 heteroatoms independently selected from nitrogen, oxygen and sulfur,where the nitrogen and sulfur heteroatoms can be optionally oxidized andthe nitrogen heteroatom can be optionally quaternized, includingmonocyclic, bicyclic, and tricyclic ring systems. The bicyclic andtricyclic ring systems may encompass a heterocycle or heteroaryl fusedto a benzene ring. The heterocycle can be attached via any heteroatom orcarbon atom. Heterocycles include heteroaryls as defined above.Representative examples of heterocycles include, but are not limited to,aziridinyl, oxiranyl, thiiranyl, triazolyl, tetrazolyl, azirinyl,diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl,oxetanyl, thietanyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl,oxazinyl, thiazinyl, diazinyl, dioxanyl, triazinyl, tetrazinyl,imidazolyl, tetrazolyl, pyrrolidinyl, isoxazolyl, furanyl, furazanyl,pyridinyl, oxazolyl, benzoxazolyl, benzisoxazolyl, thiazolyl,benzthiazolyl, thiophenyl, pyrazolyl, triazolyl, pyrimidinyl,benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl,benzoxazolyl, benzisoxazolyl, purinyl, indolyl, isoquinolinyl,quinolinyl and quinazolinyl. A heterocycle group can be unsubstituted oroptionally substituted with one or more substituents as described hereinthroughout.

Unless otherwise stated, the term “heterocycloalkyl,” by itself orcombined with other terms, represents cyclic versions of “heteroalkyl.”Additionally, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

“Lanost-7-en-26-oic acid” refers to a compound represented by followingstructure:

“Oxo” refers to the oxygen atom (O═).

“Carbonyl” refers to the group (O═C).

“Thione” refers to the sulfur atom (S═).

“Thiocarbonyl” referred to the group (S═C).

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a stereomerically pure compound having one chiral centerwill be substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. In someembodiments, a stereomerically pure compound comprises greater thanabout 80% by weight of one stereoisomer of the compound and less thanabout 20% by weight of other stereoisomers of the compound, for examplegreater than about 90% by weight of one stereoisomer of the compound andless than about 10% by weight of the other stereoisomers of thecompound, or greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, or greater than about 97% by weight of one stereoisomer ofthe compound and less than about 3% by weight of the other stereoisomersof the compound.

If there is a discrepancy between a depicted structure and a name givento that structure, the depicted structure controls. If thestereochemistry of a structure or a portion of a structure is notindicated with, for example, bold, wedged, or dashed lines, then thestructure or portion of the structure is to be interpreted asencompassing all stereoisomers of it.

“Solvate” is a form of a compound of formula I, where solvent moleculesare combined in a definite ratio as an integral part of the crystalstructure of the compound.

Depending on the structure of its referent, the phrase “pharmaceuticallyacceptable salt” denotes a pharmaceutically acceptable organic orinorganic acid or base salt of a compound of formula I. Representativepharmaceutically acceptable salts include, e.g., alkali metal salts,alkali earth salts, ammonium salts, water-soluble and water-insolublesalts, such as the acetate, amsonate(4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate,bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium,calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate,einbonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts.Furthermore, a pharmaceutically acceptable salt can have more than onecharged atom in its structure. In this instance the pharmaceuticallyacceptable salt can have multiple counterions. Accordingly, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterions.

“Synthetic compound” refers to a compound that is prepared by chemicalsynthesis or is prepared by and then isolated from cell culture. Ineither case, the compound is “purified,” in that it is in a form that isat least free of cellular debris and, preferably, is sufficiently freeof contaminants to be suitable for pharmacological use, as describedhere.

Accordingly, the present invention provides a synthetic compoundaccording to formula I or a stereoisomer, solvate, or pharmaceuticallyacceptable salt thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group.

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

It should be understood that formula I does not include(25R)-26-hydroxy-4-cholesten-3-one or lanost-7-en-26-oic acid.

In some embodiments, Q and Q′ together with the carbon atom to whichthey are attached form a carbonyl or thiocarbonyl group.

In some embodiments, R¹ is selected from the group consisting of:

In some embodiments, the invention provides a compound of formula Iwherein at least one — is a double bond. In some aspects, the compoundshave formulae Ia-1g:

In other embodiments, the invention provides a compound of formula I,wherein R² is selected from the group consisting of halo, (C₁-C₈)alkyl,and (C₁-C₈)haloalkyl. In some aspects, R² is fluoro, methyl, ortrifluoromethyl.

In still other embodiments, the invention provides a compound having theformula:

wherein R² is selected from the group consisting of fluoro, methyl, andtrifluoromethyl. In some aspects R² is methyl.

In another embodiment, n is 2. In some such aspects R² is methyl.

In some embodiments, the invention provides a compound of formula I orI′ having formulas Ia′, Ic′, Ie′, If′, and 1g′:

wherein R¹, R², and n are as defined for formula I.

In some embodiments, the invention provides a compound having thestructure:

In certain embodiments, the invention provides a compound having thestructure

In accordance with another aspect of the invention, a method is providedfor controlling the growth of a nematode by contacting it with abiologically effective amount of at least one compound of formula I or astereoisomer, solvate, or pharmaceutically acceptable salt thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group;

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

Specific compounds for controlling nematode growth include:

This invention contemplates the development of strategies againstnematodes, including but not limited to parasitic nematodes. Manyparasitic nematodes contain homologs of the orphan nuclear receptorDAF-12, which, as described above, is a key regulator of reproductivedevelopment in C. elegans. See, e.g., Siddiqui et al. (2000). Inaccordance with the present invention, therefore, ligands that areoperative in the DAF-9/DAF-12 pathway in C. elegans will affect the lifecycles of parasitic nematodes similarly. The inventors have discoveredin Anycolostoma ceylancium, for example, a DAF-12 homolog that isactivated by DAF-12 ligands, identified pursuant to this invention. Inturn, co-transfection of DAF-9, using the assays described below, causesactivation of this DAF-12 homolog when cells are contacted withlathosterone.

Illustrative of parasitic nematodes in this regard are members of anorder selected from Strongylida, Rhabditida, Ascaridida, Spirurida,Oxyurida, Enoplida, Tylenchida, and Dorylaimida nematode orders. Otherexamples of parasitic nematode include members of a genus selected fromHaemonchus, Oestertagia, Trichostrongylus, Cooperia, Diclyocaulus,Strongylus, Oesophagostomum, Syngamus, Nematodirus, Heligmosomoides,Nippostrongylus, Metastrongylus, Angiostrongylus, Acyclostoma, Necator,Uncinaria, Bunostomum, Strongyloides, Steinernema, Ascaris, Parascaris,Toxocara, Toxascaris, Baylisascaris, Anisakis, Pseudoterranova,Heterakis, Wuchereria, Brugia, Onchocerca, Dirofilaria, Loa, Thelazia,Dracunculus, Gnathostoma, Enterobius, Oxyuris, Syphacia, Trichinella,Trichuris, Capillaria, Globodera, Heterodera, Meloidogyne, Anguina,Ditylenchus, Hirschmanniella, Naccobus, Pratylenchus, Radopholus,Criconema, Tylenchulus, Paratylenchus, Aphelenchus, Bursaphelenchus,Longidorus, Xiphinema, Trichodorus, and Paratrichodorus. Specificparasites include Strongyloides Stercoralis and Acycolostoma ceylanicum.

In another embodiment, the invention provides a method for reducing orpreventing nematode infestation of a plant that is infested orsusceptible to infestation by a nematode population, comprisingadministering to the plant a biologically effective amount of at leastone compound according to formula I or a stereoisomer, solvate, orpharmaceutically acceptable salt thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group.

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl (C₁-C₆)alkyl,heteroaryl (C₁-C₆)alkyl, aryl (C₁-C₆)alkyl, NRR′, oxo, thione, OR, andSR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

In some aspects of the inventive method, administering comprisestreating with the compound, soil in which the plant is grown. In otheraspects, the administering comprises treating, with the compound, a seedfrom which the plant is to be grown.

In another embodiment, the invention provides a method of reducing orpreventing a nematode infestation in a mammal, comprising administeringto the mammal a therapeutically effective amount of at least onecompound according to formula I or a stereoisomer, solvate, orpharmaceutically acceptable salt thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group;

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound according to formula I or a stereoisomer, solvate, orpharmaceutically acceptable salt thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group.

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

In some embodiments, the composition contains a compound is selectedfrom the group consisting of:

In general, the compounds of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound, as the active ingredient, will depend uponnumerous factors, such as the severity of the disease to be treated, theage and relative health of the subject, the potency of the compoundused, the route and form of administration, and other factors. The drugcan be administered more than once a day, and preferably once or twice aday. All of these factors are within the skill of the attendingclinician.

Therapeutically effective amounts of compounds of formula I may rangefrom approximately 0.05 to 50 mg per kilogram body weight of therecipient per day; preferably about 0.1-25 mg/kg/day, more preferablyfrom about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kgperson, the dosage range would most preferably be about 35-70 mg perday. Modes of administration include oral, systemic (e.g., transdermal,intranasal, or suppository), intrathecal, and parenteral (e.g.,intramuscular, intravenous, or subcutaneous).

The choice of formulation depends on various factors such as the mode ofdrug administration and bioavailability of the drug substance. Fordelivery via inhalation the compound can be formulated as liquidsolution, suspensions, aerosol propellants or dry powder and loaded intoa suitable dispenser for administration. There are several types ofpharmaceutical inhalation devices-nebulizer inhalers, metered doseinhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices producea stream of high velocity air that causes the therapeutic agents (whichare formulated in a liquid form) to spray as a mist that is carried intothe patient's respiratory tract. MDI's typically are formulationpackaged with a compressed gas. Upon actuation, the device discharges ameasured amount of therapeutic agent by compressed gas, thus affording areliable method of administering a set amount of agent. DPI dispensestherapeutic agents in the form of a free flowing powder that can bedispersed in the patient's inspiratory air-stream during breathing bythe device. In order to achieve a free flowing powder, the therapeuticagent is formulated with an excipient such as lactose. A measured amountof the therapeutic agent is stored in a capsule form and is dispensedwith each actuation.

In general, the compositions of the invention are comprised of acompound of formula I in combination with at least one pharmaceuticallyacceptable carrier or excipient. Acceptable excipients are nontoxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe compound of formula I. Such excipient may be any solid, liquid,semi-solid or, in the case of an aerosol composition, gaseous excipientthat is generally available to the field.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc. Other suitable pharmaceutical excipients and theirformulations are described in REMINGTON'S PHARMACEUTICAL SCIENCES,18^(th) ed. (Mack Publishing Co., 1990).

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt% of a compound of formula I based on the total formulation, with thebalance being one or more suitable pharmaceutical excipients.Preferably, the compound is present at a level of about 1-80 wt %.

In another embodiment the invention provides a culture system forpreparing a compound having formula I or a stereoisomer thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group.

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

R² is a substituent on one or more carbon atoms in rings A, B, C, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

The system comprises cells expressing DAF-9 and media containing asteroid precursor of formula I wherein R¹ is:

wherein R¹ is optionally substituted with 1 to 8 substituents, each ofwhich is independently selected from the group consisting of halo,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy(C₁-C₄)alkyl,(C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR; and whereinR³ and R^(3′) are independently hydrogen or hydroxy or R³ and R^(3′)together with the carbon atom to they are attached form a carbonylgroup.

In some embodiments, the compound produced by the culture is selectedfrom the group consisting of

and the steroid precursor is selected from the group consisting of

The compounds of formula I can be produced by means of an insect cellexpression system, involving, for example, baculovirus-mediated genetransfer to Sf9 insect cells grown in liquid suspension. Such abaculovirus expression system is readily scaled to industrial level andis used extensively for protein expression. See, e.g., S. R. Hood et al.(1996).

In one such method, Sf9 cells are infected with baculovirus expressingDAF-9 and the human cytochrome P450 oxidoreductase. Aftersupplementation with the appropriate heme precursors and Fe supplements,infected cells are grown for 60 hours in the presence of DAF-9substrates. Certain DAF-9 substrates are commercially available(4-cholesten-3-one and 4,6-cholestadiene-3-one) while others can besynthesized from commercially available starting materials usingcholesterol oxidase to produce 4,7-cholestadiene-3-one from7-dehydrocholesterol or Dess Martin to produce lathosterone fromlathosterol. After about 60 hours, cells and media are harvested and thesubstrate and products are recovered by extracting with organicsolvents. Alternatively, membrane-bound enzyme can be obtained bysub-cellular fractionation, and the resulting membranes are incubated ina cell-free system with appropriate reducing agents and substrates.

This technique is powerful because it also may be used to reconstitutethe entire pathway from cholesterol. In addition, it is possible thatenzymes in C. elegans modify DAF-12 ligands to convert them intoantagonists or partial agonists. Such enzymes could be used in thissystem for enzymatic synthesis as well.

The compounds of formula I also can be synthesized by chemical methods.For example, various compounds of formula I may be synthesized, viaknown chemical transformations, from diosgenin or yamogenin:

Thus, diosgenin is converted to 25R-cholest-5-ene-3β,26-diol3β,26-di-TBDMS ether (1), pursuant to the protocol of Kim et al. (1989),which entails, seriatim, a Clemmensen reduction, a 3,26-diprotectionwith t-butyldimethylsilylchloride, an oxidation to the 16-keto form, anda deoxygenation of the 16-keto (reactions “a” in Scheme I below).

Schemes I-III feature protecting groups at the 3- and 26-hydroxyl groupsas t-butyldimethylsilyl groups (TBDMS), yet in some routes these groupsmust be replaced with acetate or other protecting groups for specificsteps as is standard procedure in the art. In the work immediatelybelow, compounds 3, 4, and 5 are also or exclusively described as the3,26-diacetates.

Via a procedure adapted from Yu et al. (2002), 1 is subject tohydroboration, yielding 5α-25R-cholest-3β,6β,26-triol 3β,26-di-TBDMSether (2, reactions “b”), and subsequent oxidation of 2 provides5α-25R-cholest-3β,26-diol-6-one 3β,26-di-TBDMS ether (3, reactions “c”).The procedure is as follows:

To a stirred solution of 1 (1.62 g, 2.57 mmol) in THF (30 mL) is addedneat BH₃.Me₂S (0.7 mL, 7.71 mmol) under positive pressure of argon, andthe reaction mixture is stirred for 12 hours at room temperature. Afterquenching by drop-wise addition of water, an aqueous solution of NaOH(30%, 20 ml) and then H₂O₂ (30%, 20 mL) is added, and stirring continuesovernight. After an adjustment of pH to 7 with dilute aqueous HCl, thesolution is poured into saturated aqueous NaCl (40 mL) and extractedwith diethyl ether (60 mL×4). The organic layer is concentrated invacuo, to afford a residue (1.55 g of crude 2). The crude product isdissolved in 30 mL of dichloromethane, and Dess-Martin periodinane (1.11g, 2.61 mmol) is added slowly, with stirring. After stirring at roomtemperature for 5 hours, the suspension is filtered and concentrated invacuo. The residue is purified by flash chromatography on silica gelwith 20:1 petroleum ether/ethyl acetate, to provide 3 (1.21 g, 73% yieldfrom 1) as white solid.

Compound 3 then is converted to its diacetate (aqueous HF, followed byacetic anhydride in pyridine) and subjected to bromination, yielding5α-25R-cholest-7-bromo-3β,26-diol-6-one 3β,26-diacetate (4, reaction“d”), via the following procedure, adapted from Yu et al. (2002): asolution of the diacetate of 3 (0.05 g, 0.099 mmol) in 5 mL dry THF wastreated with pyridinium tribromide (0.083, 0.261 mmol) at 0° C. andbrought to room temperature. After stirring at room temperature for 1.5h, saturated Na₂S₂O₃ solution (2 mL) was added to quench the reaction.The product was extracted twice into 10 mL of diethyl ether, whichwashed with 20 mL each water and brine. The residue was purified byflash chromatography, eluting with 14% ethyl acetate/hexanes to afford 4(0.045 g, 78%) as a white solid.

Elimination of 4 to 5α-25R-cholest-7-ene-3β,26-diol-6-one3β,26-diacetate (5, reaction “e”) was effected by a procedure adaptedfrom Jiang et al. (2003), as follows: a mixture of 4 (0.012 g, 0.02mmol), Li₂CO₃ (0.0003 g, 0.004 mmol) and LiBr (0.002 g, 0.02 mmol) in 5mL DMF is heated at 125° C. under nitrogen for 24 hours. After coolingto room temperature, ethyl acetate (30 mL) is added, and the organiclayer is washed with water until neutral pH, then dried over Na₂SO₄. Thesolvent is removed in vacuo, and the residue is purified by flashchromatography on silica gel (eluting with 2:3 ethyl acetate/hexanes) togive 5 (0.008 g, 77%) as a white solid.

Compound 5 may be converted into active DAF-12 ligands with a variety ofchemical configurations in the A/B ring system and an alcohol or acid atthe 26 position. Scheme II describes chemistry, completed or prophetic,for obtaining desired configurations in the A/B ring system. Scheme IIIdescribes a common set of reactions applied to the products of Scheme IIor to compound 5 itself to complete the synthesis of DAF-12 ligands byremoving protecting groups and adjusting the oxidation states of carbonatoms 3 and 26. For example compound 5 is reduced anddeoxygenated/isomerized to compound 7 according to the procedures ofHaag et al. (1988) and Zheng et al. (2003) (reactions “f”). Severalapproaches as described below are useful to convert compound 5 tocompound 6, in which the A/B ring system is configured according to oneof the endogenous DAF-12 ligands identified in this invention. Severalintermediates in the synthesis of the final compounds may also beagonists or antagonists (pure, partial, and inverse) for DAF-12 andrelated receptors in other nematode species.

Compound 5 (0.020 g, 0.034 mmol) was dissolved in a mixture of drydichloromethane (1 ml) and dry THF (2 ml). After addition of methanol (3ml), CeCl₃.7H₂O (0.014 g, 0.037 mmol) was added followed by NaBH₄ (0.002g, 0.05 mmol) at 0° C., and the solution was then allowed to reach roomtemperature slowly with stirring. After 1 h, the reaction was dilutedwith chloroform (5 ml) and quenched with 10 mL water. The organic phasewashed with 10 mL each water and brine, and the product was purified byflash chromatography, eluting with 22% ethyl acetate in hexanes toafford a mixture of the 6-alcohols (0.018 g, 90%) as a white solid. Themixture of 6-alcohols (0.0042 g, 0.008 mmol) was dissolved in methylenechloride (5 ml) and cooled to 0° C. Triethylsilane (7.6 μl, 0.048 mmol)was added to the solution, and then boron trifluoride etherate (10 μl,0.08 mmol) was added dropwise. After the mixture was stirred for 15 min,10% sodium carbonate (2 ml) was added. The aqueous layer was extractedwith 10 mL methylene chloride. The combined methylene chloride extractswere washed with 10 mL brine, dried over anhydrous Na₂SO₄, andevaporated in vacuo. The residue was purified by flash chromatography,and the product 7 eluted with 14% ethyl acetate in hexanes as a whitesolid (0.0038 g, 94%).

Several synthetic routes from compound 5 to compound 6 are describedbelow, either as a single reaction sequence (reaction “g”):

-   -   1) Modified Wolff-Kishner reduction of ketone 5 (or TBDMS        analog) with hydrazine hydrochloride/hydrazine hydrate and        potassium hydroxide;    -   2) Conversion of ketone 5 to the thioketal with        ethanedithiol/BF₃ etherate and reduction with Raney nickel;        or as multiple-step routes (triple arrows):    -   1) Reduction of 6-ketone 5 to the 6-alcohols as described above,        followed by conversion to 6-bromide with either PBr₃ (or        1,2-phenylene phosphorobromidite), or CBr₄/triphenylphosphine,        followed by        -   a) reduction using hydrogen gas/catalyst, zinc dust, sodium            or lithium metal.        -   b) formation of Grignard reagent with Mg turnings and            quenching with ethanol.    -   2) Reduction of 6-ketone 5 to the 6-alcohols as described above,        followed by conversion to 6-iodide with        iodine/triphenylphosphine/imidazole followed by reduction with        SmI₂.    -   3) Reduction of 6-ketone 5 to the 6-alcohols as described above,        followed by elimination using methylsulfonyl        chloride/triethylamine to the 5,7-diene; selective 5α-reduction        of the delta-5 olefin with H₂ gas and Raney nickel or other        suitable catalyst.

Scheme III depicts a general route from the diprotected intermediates tothe DAF-12 ligands. Thus, deprotection of the TBDMS (or acetate) groupsyields the 3,26-diols (e.g., compound 8). Oxidation with CrO₃ (Jonesreagent) in acetone gives the 3-keto-26-acids directly (Ia, reactions“h”). The 3-keto-26-alcohols (Ib) may be prepared by selectiveprotection of the 26-hydroxyl with TBDMS chloride in methylene chloride(yielding compound 9, reactions “i”), oxidation with Dess-Martinperiodinate or pyridinium dichromate (yielding compound 10, reactions“j”), followed by deprotection with aqueous HF (reaction “k”). The3-hydroxy-26-acids (not shown) are prepared by acetylation of the26-TBDMS ether with acetic anhydride in pyridine, deprotection/oxidationwith Jones reagent, and deprotection of the 3-hydroxyl with aqueoussodium methoxide.

These routes afford several compounds that are 25R isomers fromdiosgenin and 25S isomers from yamogenin. A scheme for generating 25Sisomers from 25R,26-acids (and vice-versa) can be carried out asfollows: the 25R acids are converted to their acyl chlorides with(COCl)₂ in dimethylformamide. The acyl chlorides are treated with(−)-carenediol and dimethylaminopyridine in THF to yield the carenediolesters. The 25 position is isomerized with potassium t-butoxide int-butanol at room temperature to the 25R/S mixture. The isomers areseparated by reverse phase HPLC and isolated. Hydrolysis of the esterswith LiOH in aqueous THF yields the individual acids.

According to another of its aspects, the present invention provides amethodology for identifying a modulator of DAF-12 or a DAF-12 homolog.In general terms, the inventive method employs a cell line, typicallymammalian or insect, that is characterized by the presence of anheterologous polynucleotide sequence coding for (i) a polypeptide thatcontains the full length DAF-12 or ligand-binding domain of DAF-12 (“theDAF-12 polypeptide”) and (ii) a DAF-12-responsive reporter, respectively(see below). Cells of such a line are brought into contact with putativemodulators, as may be presented in the form of a library of smallmolecules or peptides, in order to screen for compounds that bind to theDAF-12 domain. In a variation of this approach, the aforementioned cellline is engineered to express DAF-9 as well, and the resultant cells areused to screen for a compound that DAF-9 metabolizes into DAF-12 ligand,which can activate DAF-12 to prevent dauer formation or to driverecovery from dauer.

In accordance with the invention, therefore, an assay can be performedto identify antagonists of either the enzyme, DAF-9, or the receptor,DAF-12, with essentially the same outcome in each instance. Moreover,the assay can be adapted to effect high-throughput compound screens,which may be automated by means of commercially available roboticscreening systems, such as CRS Ultra High Throughput Screening System, aproduct of the Thermo Electron Corporation (Waltham, Mass.).

By way of illustration of such an assay, one can employ conventionalrecombinant DNA technology, along the lines described by Makishima etal. (1999), to fuse the modular ligand-binding domain of DAF-12 to theC-terminus of the DNA binding domain of the yeast transcription factor,GAL4. The resulting fusion protein can bind specifically to a GAL4 DNAresponse element in a GAL4-responsive reporter gene plasmid, encoding adetectable marker such as luciferase, whereby receptor activity can bemonitored after treatment with putative ligand.

Alternatively, the full-length DAF-12 receptor is used, along with aDAF-12-responsive reporter that also contains one or more DAF-12 DNAresponse elements, as described, for example, by Shostak et al. (2004).Although similar in certain respects to the foregoing illustration, thisassay mode employs a DAF-12-specific reporter gene rather than aGAL4-specific reporter gene. The specificity resides in the DAF-12 DNAresponse element(s), which were identified within fragments of C.elegans genomic DNA in screens for genomic DNA fragments that bind toDAF-12 in vitro. When linked to a reporter gene such as GFP, these DNAfragments drive expression of GFP in vivo in a DAF-12-dependent manner.With these endogenous DAF-12 response elements, the full-length DAF-12receptor, containing both natural DNA binding domain and ligand bindingdomain, must be used in the screen as opposed to the GAL4-hybridpolypeptide. It is possible, however, that these response elements maybe regulated by mammalian nuclear receptors; hence, activation of thereporter would need to be tested in absence of DAF-12, to control forthe presence of competing nuclear receptors that are endogenous to thecell lines used.

The above-discussed GAL4-responsive luciferase reporter is preferred,because GAL4 is specific to yeast and its DNA response elements arebound only by GAL4. Accordingly, there is no potential for interferencefrom nuclear receptors that may be endogenous to the cell line.

As noted, compound libraries can be screened for agonists/antagonists ofDAF-12, once cells are provided that harbor the appropriate receptor andreporter plasmids. It has not been possible to identify antagonists ofDAF-12 heretofore, for the simple fact that no one had identified anagonist of DAF-12. Pursuant to the present invention, one can screen forcompounds that antagonize activation of DAF-12 by one or more of theDAF-12 ligands described presently, preferably employing a ligandconcentration in the range between about EC₅₀ and about EC₈₀, e.g.,approximately 200 nM to 4 μM.

Another approach, according to the invention, is to attack both DAF-9and DAF-12 together. This can be done by screening for compounds thatinhibit the ability of DAF-9 to metabolize its substrates into ligandsof DAF-12. This type of antagonist screen would not be possible withoutthe identification of DAF-9 substrates, per the present inventors'discovery. Such a screen allows for detection of compounds thatantagonize reporter gene activity at the level of DAF-9 ligandproduction or DAF-12 activation. The primary target, DAF-9 versusDAF-12, can be determined by removing DAF-9 from the assay and testingthe “hit” for antagonism of the DAF-12 ligands described here. Othercontrols systems may be employed to test for DAF-9 specificity, such asa similar co-transfection system, described by Cheng et al. (2003), thatinvolves cytochrome P450 2R1-mediated activation of the Vitamin Dreceptor. Briefly, 1αOH vitamin D₃ is supplied to cells co-transfectedwith both CYP2R1 and the vitamin D receptor. The conversion of1αOHvitamin D₃ to 1α,25-dihydroxyvitamin D₃ is monitored throughactivation of the Vitamin D receptor. Lack of inhibition of this systemby candidate DAF-9 antagonists would indicate the antagonist propertieswere specific to the DAF-9 P450 and not a general inhibitor of mammalianP450s.

The DAF-12 ligand and DAF-9 substrates used in the assay includecompounds of formula I

wherein R² is a substituent on one or more carbon atoms in rings A, B,C, and D and, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl (C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies. For theDAF-12 ligand, R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.

For the DAF-9 substrate, R¹ is

wherein R¹ is optionally substituted with 1 to 8 substituents, each ofwhich is independently selected from the group consisting of halo,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy(C₁-C₄)alkyl,(C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR; and whereinR³ and R^(3′) are independently hydrogen or hydroxy or R³ and R^(3′)together with the carbon atom to they are attached form a carbonylgroup.

In some embodiments of the assay, the DAF-12 ligand is selected from thegroup consisting of

In some embodiments of the assay, the DAF-9 substrate is selected fromthe group consisting of

The assay methodology of the present invention can accommodate any of arange of detectable markers, including but not limited to a detectablepolypeptide, such as a fluorescent polypeptide, a chemiluminescentpolypeptide, an epitope tag, and an enzyme. Thus, a suitable marker canbe selected from among human growth hormone, luciferase, chloramphenicolacetyl transferase, xanthine-guanine phosphoribosyl transferase, andβ-galactosidase, or a variant of any of these.

According to the invention, the category of DAF-12 modulators includescompounds that act as agonists or antagonists. Screening for agonistsentails testing for activation of DAF-12 relative to a vehicle control,using ligands, such as those described here, as a positive control. Onthe other hand, an antagonist inhibits or reduces (partiallyantagonizes) the activation of DAF-12 by a DAF-12 ligand, through anability to bind DAF-12, competitively or non-competitively. The categoryof DAF-12 modulators also encompasses inverse antagonists, which bindelsewhere than the ligand-binding pocket of DAF-12, to stabilize theinactive conformation of the receptor.

Compounds that modulate DAF-12 through activation can be identified in ascreen, pursuant to the present invention. Such compounds may be morepotent or efficacious than the endogenous DAF-12 ligand, therebyrepresenting not only a potent activator of DAF-12 but also a new toolto be used in the study of nematode physiology.

In another embodiment, therefore, the invention encompasses a kit foridentifying DAF-12 modulators, comprising a compound of formula I or astereoisomer thereof:

wherein one of Q or Q′ is OH or SH and the other of Q or Q′ is hydrogen,or Q and Q′ together with the carbon atom to which they are attachedform a carbonyl or thiocarbonyl group.

R¹ is selected from the group consisting of:

wherein each instance of ═ independently is a single bond, a doublebond, or a triple bond; and wherein R¹ is optionally substituted with 1to 8 substituents, each of which is independently selected from thegroup consisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)Cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, OR, and SR.R² is a substituent on one or more carbon atoms in rings A, B, and Dand, in each instance, is independently selected from the groupconsisting of halo, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)haloalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)Cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, NRR′, oxo, thione, OR, and SR.

R and R′ are independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Variable n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Each instance of — independently is a single bond or a double bond;wherein one of — is optionally absent so that one of rings A, B, C or Dis no longer a cyclic ring or part of a cyclic ring, and wherein when —is absent, the two resultant terminal carbon atoms are substituted withone or more hydrogen atoms to satisfy the carbon atom valencies.

In one embodiment, the kit contains a cDNA for a DAF-12 polypeptidecomprised of a ligand binding domain or a portion thereof and a DNAbinding domain or portion thereof, in a DNA plasmid that allowsexpression of said cDNA in a mammalian cell line. In some aspects, theplasmid is a pCMX-GAL4-DAF12 plasmid.

In another embodiment, the kit contains a cDNA encoding DAF9 polypeptideor modified version thereof with equivalent enzymatic activity in a DNAplasmid that allows expression of said cDNA in a mammalian cell line,such as pCMX-DAF9 plasmid.

In other embodiments, the kit contains a reporter gene DNA plasmid whoseexpression is driven by activated DAF-12 polypeptide. In some aspectsthe plasmid is a pMH100x-4-luc plasmid that has the firefly luciferasecDNA adjacent to the thymidine kinase promoter and four copies of theGAL4-DNA response element.

In still other embodiments, the kit contains reagents for detecting theexpression of the reporter gene, such as the Dual-Luciferase® ReporterAssay System marketed by Promega, Inc. (Madison, Wis.).

In another embodiment, the kit contains a suitable cell line forexpression and assay of DAF-12. In some aspects the cell line is a humanembryonic kidney (HEK-293) cell line (American Type Culture Collection,Catalog #CRL-1573, Manassas, Va.), or a cell line that stablyincorporates and expresses said DAF-12 cDNA in its genome.

In other embodiments, the kit contains reagents for inserting thedesired DNA plasmids into the cell line, such as the FuGENE6® reagent(Roche Biochemicals, Indianapolis, Ind.).

The following examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in practicing theinvention. These examples are in no way to be considered to limit thescope of the invention.

EXAMPLES Chemical Reagents

4-Cholesten-3-one, lathosterol, 20S-hydroxycholesterol,22S-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 3-keto-lithicholic acid, 7-keto-lithocholic acid,12-keto-lithocholic acid were purchased from Steraloids, Inc. (Newport,R.I.). Lophenol, (25S),26-hydroxycholesterol, and(25R),26-hydroxycholesterol were purchased from Research Plus(Manasquan, N.J.). Unless otherwise noted, all other reagents werepurchased from Sigma. All sterols were dissolved in ethanol and storedat −20° C.

Sterol Synthesis

The 3-keto-Δ⁴-oxysterol derivatives in FIG. 3A were generated withcholesterol oxidase and catalase as described (Zhang et al. 2001). The3-keto sterols of lathosterol and lophenol (lathosterone and lophenone)were generated by chemical oxidation with Dess-Martin periodinanereagent. Briefly, 2.5 molar equivalents of Dess-Martin reagent werereacted with one equivalent of each sterol at room temperature.Reactions were monitored by TLC using 95:5 hexane:ethylacetate.Lophenone and lathosterone were purified from starting materials usingsilica chromatography with 95:5 hexane:ethylacetate. Structures wereconfirmed by ¹³C and ¹H-NMR. To oxidize (25R) and(25S),26-hydroxy-4-cholesten-3-one into C-26 acids, Jones reagent (0.14mL, 0.15 mmol) was added slowly and dropwise to a stirred solution ofthe alcohol (12 mg, 0.03 mmol) in acetone (4 mL) at 0° C. After stirringfor 1 hour, the reaction was quenched with isopropanol, and the productextracted with diethyl ether. The organic phase washed with saturatedNaHCO₃, dried over solid Na₂SO₄, filtered, and concentrated in vacuo.Crude extracts were chromatographed on silica gel, and the product (10mg, 90% yield) eluted with 40% ethyl acetate in hexane. All structureswere confirmed by MS, UV spectra, and ¹³C- and ¹H-NMR (FIG. 11).

Alternate Syntheses of Compounds of Formula I

The following examples illustrate synthetic routes other than thosedepicted in Schemes I-III above by which to make compounds of Formula I.

25(R)-cholest-5-en-3β,26-diacetate (II)

To a solution of 25(R)-cholest-5-en-3β,26-diol (1.31 g, 3.25 mmol,prepared as described in the procedure above for 1; Scheme 1, reaction“a”) in dry pyridine (15 mL), were added acetic anhydride (3 mL) andN,N-dimethylaminopyridine (4 mg, 0.027 mmol). The mixture was stirred atroom temperature under nitrogen for 18 h. Water (5 mL) was added, andthe product was extracted with ethyl acetate. The organic layer washedwith NaHCO₃, then water, and dried over Na₂SO₄. The solvent wasevaporated to dryness. The crude product was subjected to MPLC (silicagel 60-200 mesh, 1.0×12 inch column, elution with ethyl acetate-hexanes6:100) to give II (1.21 g, 76%).

25(R)-7-bromo-cholest-5-ene-3β,26-diol-3,26-diacetate (III)

To a solution of II (0.081 g, 0.166 mmol) in benzene-hexane 1:1(10 mL)was added N,N-dibromohydantoin (0.023 g, 0.083 mmol). The mixture wasrefluxed under nitrogen for 10 min in a preheated oil bath at 100° C.and then placed in an ice bath to cool. Insoluble material was removedby suction filtration, followed by evaporation of the filtrate to ayellow solid. The crude product can be purified and the unreactedstarting material recovered/recycled, or crude III can be used directlyin the next reaction.

25(R)-cholest-5,7-diene-3β,26-diol-3β,26-diacetate (IV)

To a solution of the yellow solid (crude III) in anhydroustetrahydrofuran (8 mL) was added tetrabutylammonium bromide (0.016 g,0.049 mmol). The resulting solution was stirred for 75 min undernitrogen at room temperature and then treated with tetrabutylammoniumfluoride (0.35 ml, 1M solution in THF, 0.35 mmol, 2.1equivalents). The resultant dark brown solution was stirred for anadditional 50 min, followed by rotatory evaporation to a brown solid.The residue was dissolved in ethyl acetate (100 mL), washed with water(3×25 mL), and dried over Na₂SO₄. Evaporation of solvent gave crude IV(0.1 g), which was subjected to MPLC on AgNO₃ impregnated silica toafford 0.02 g (25%). Unreacted II is recovered following MPLC.

25(R)-cholest-7-ene-3β,26-diol-3β,26-diacetate (V)

To a solution of IV (0.015 g, 0.03 mmol) in freshly distilled ethylacetate (7 mL) was added platinum (IV) oxide catalyst (Aldrich P345).The mixture was stirred at ambient temperature for 2 h in 1 atm ofhydrogen gas. After completion of reaction, the reaction mixture wasfiltered through a small plug of silica gel and purified on silica gel60-200 mesh by standard column chromatography, eluting with 6% ethylacetate in hexanes to afford 0.014 g (89%).

25(R)-cholest-7-ene-3β,26-diol (VI)

To a solution of diacetate V (0.012 g, 0.024 mmol) in moist methanol wasadded 200 mg of KOH. After refluxing for 4 h, the reaction mixture wasneutralized with dilute HCl, and the methanol was removed under reducedpressure. The residue was dissolved in diethyl ether, which washed with2% NaHCO₃ and dried over Na₂SO₄. After evaporation under reducedpressure, the product was chromatographed on silica gel 60-200 mesh bystandard column chromatography and eluted with 22% ethyl acetate inhexanes to afford 0.009 g VI (90%).

25(R)-cholest-7-ene-26-al-3-one (VII)

To a solution of diol VI (0.008 g, 0.019 mmol) in dry dichloromethane (4mL) at ambient temperature was added Dess-Martin periodinane (0.017 g,0.04 mmol). After 4 h, the reaction was quenched with saturated Na₂S₂O₃solution and diluted with 100 mL dichloromethane. The organic phasewashed with 3×25 mL saturated Na₂S₂O₃ solution and brine, then driedover Na₂SO₄. After evaporation under reduced pressure, the residue waschromatographed on silica gel 60-200 mesh by standard columnchromatography, and the product eluted with 6% ethyl acetate in hexanesto afford 0.007 g VII (88%).

25(R)-cholest-7-ene-3-one-26-oic acid [25(R)-Δ⁷-dafachronic acid] (VIII)

To a stirred solution of aldehyde VII (0.006 g, 0.015 mmol) in methanol(3 mL) was added isobutene (0.016 μl, 0.15 mmol), then 0.0028 g of 1.25M NaClO₂ in 20% NaH₂PO₄,** and stirring was continued at roomtemperature for 1 h. The reaction was quenched with saturated NH₄Clsolution and diluted with 100 mL of diethyl ether. The organic phase waswashed with 3×25 mL 2% NaHCO₃ solution, then brine, and dried overNa₂SO₄. After evaporation under reduced pressure, the residue waschromatographed on silica gel 60-200 mesh by standard columnchromatography, and the product eluted with 32% ethyl acetate in hexanesto afford 0.005 g VIII (80%).

**1.25 M NaClO₂ in 20% NaH₂PO₄ was prepared by adding 0.57 g NaClO₂ to a20% solution of NaH₂PO₄ in H₂O (1 g NaH₂PO₄+5 mL H₂O)

The following procedures can be adapted to compounds of Formula I inconverting 25(R)-cholestenoic acids and precursors to their25(S)-isomers.

Isomerization of 25(R)-cholest-7-ene-26-al-3-one (VII) to25(R,S)-cholest-7-ene-26-al-3-one (VIIa)

(25R)-Cholest-7-ene-3-keto, 26-aldehyde

To epimerize the stereochemistry at carbon-25,25(R)-aldehyde VII (0.007g, 0.017 mmol) was dissolved in THF (5 mL), and1,8-diazabicyclo[5.4.0]undec-7-ene (0.3 mL) was added, followed bystirring at ambient temperature for 48 h. The THF was evaporated underreduced pressure, and the residue was dissolved in ethyl acetate, whichwashed with brine and dried over Na₂SO₄. After evaporation under reducedpressure, the residue was chromatographed on silica gel 60-200 mesh bystandard column chromatography, and the product eluted with 6% ethylacetate in hexanes to afford 0.006 g VIIa (85%). ¹H-NMR showsepimerization of aldehyde proton (two doublets).

The resulting aldehydes are oxidized to epimeric acids VIII or reducedto epimeric alcohols VI. The epimers can be resolved by high-performanceliquid chromatography before or after derivatization as chiral esterswith chiral auxiliaries, such as (−)-carenediol or Mosher ester.

The reactions below are described with model compound and precursor25(R)-cholest-5-ene-3β,16β,26-triol (IX), which contains an extra16-hydroxyl group relative to compounds of Formula I. The procedurestherefore can be carried out to make compounds of this invention.

25(R)-cholest-5-ene-3β,16β-diol-26-tosylate (X)

To a solution of triol IX (0.2 g, 0.47 mmol) in pyridine (4 mL) wasadded slowly p-toluenesulfonyl chloride (0.1 g, 0.52 mmol) at −10° C.,and the reaction was stirred at room temperature for 24 h. Aftercompletion of reaction, ether (100 mL) was added, and the organic phasewashed with saturated CuSO₄ (3×25 mL), dried over Na₂SO₄, andevaporated. The residue was purified by column chromatography withsilica gel 60-200 mesh using 22% ethyl acetate in hexanes, whichafforded X (0.16 g, 60%).

25(R)-26-iodo-cholest-5-ene-3β,16β-diol (XI)

A mixture of tosylate X (0.15 g, 0.26 mmol), acetone (6 mL, freshlypurified with KMnO₄ and distilled from K₂CO₃) and NaI (0.14 g, 0.96mmol) was stirred in the dark at 80° C. for 6 h. After completion ofreaction, ethyl acetate (100 mL) was added, and the organic phase washedwith water (3×25 mL), dried over NaSO₄, and evaporated to dryness. Theresidue was purified by column chromatography with silica gel 60-200mesh using 20% ethyl acetate in hexanes, affording iodide XI (0.11 g,80%).

25(R)-cholest-5,25(26)-diene-3β,16β-diol (XII)

To a stirred solution of iodide XI (0.14 g, 0.26 mmol) in dry pyridine(4 mL) was added under argon silver fluoride (0.065 g, 0.52 mmol). Thereaction mixture was stirred at ambient temperature for 12 h and thendiluted with diethyl ether (100 mL), which washed with a saturatedsolution of CuSO₄ (3×25 mL) and dried over Na₂SO₄. After evaporation ofthe solvent, the residue was purified by column chromatography withsilica gel 60-200 mesh using 18% ethyl acetate in hexanes to afforddiene XII (80 mg, 75%).

The above diene can be regioselectively and stereoselectively convertedto the 25(S) alcohol by hydroboration with (−)-diisopinocamphenylboraneand oxidative work up with H₂O₂. The steps described above can be usedto convert II to its 25(S) isomer. The synthetic methodology can be usedto convert II to the 25(S)3-keto-7,(5α)-cholestenoic acid VIII.

Alternatively, intermediate compounds can be subjected to Oppenauroxidation/isomerization, using aluminum isopropoxide andN,N-dimethylamino pyridinone, to make 25(R)- or 25(S)3-keto-4-cholestenoic acids.

Nematode and Bacterial Strains

Worms were grown on NGM agar seeded with OP50 bacteria at 20° C. unlessnoted otherwise (Brenner, 1974). Strains used were: daf-9(dh6) dhEx24(containing the cosmid T13C5 and pTG96 (sur-5::gfp)), daf-9(rh50),daf-12(rh273), and daf-12(rh61), daf-2(e1368), daf-2(e1370) daf-7(m62)and N2.

Plasmids

Inserts of the various cDNAs described below were cloned in to mammalianexpression plasmids and their derivatives containing the VP16 activationdomain or GAL4 DNA binding domain as described (Umesono et al., 1991;Willy et al., 1995). Full length cDNAs for NHR-8 (Genbank NM_(—)171382,wormbase F33D4.1a), NHR-23 (C01H6.5b), DAF-9 (Genbank NM_(—)171699,wormbase T13C5.1b), and DIN-1S (wormbase F07A11.6d), were obtained byRT-PCR from mixed stage or L2/L3 staged animals. pCMV-hCYP27A1,pCMV-mCYP27A1, and pCMV-adrenodoxin were gifts from David Russell. HumanP450 oxidoreductase cDNA was purchased from Open Biosystems. GAL4fusions were generated based on the following amino acid sequences:184-754aa (DAF-12), 92-561aa (NHR-8), 78-361aa (NHR-23), 2-567aa(DIN-1S). VP16-DAF-12 fusion was generated using 2-754aa of DAF-12. TheR564C and R564H mutants of DAF-12 were generated by site directedmutagenesis. The lit-1K-tk-luc reporter was generated by inserting the4.2 genomic fragment from pODLO_(—)82 into the reporter plasmid tk-luc.Baculovirus expression plasmids containing DAF-9 and hOR were createdusing the pFastBac Dual system (Invitrogen).

Cell Culture and Cotransfection Assay

Cotransfection assays in HEK293 cells were performed as previouslydescribed using a 96-well format (Makishima et al., 1999). DNA wasdelivered to cells containing 50 ng of luciferase reporter, 20 ngCMX-β-galactosidase reporter, 15 ng of CMX receptor expression plasmid,and enough control plasmid to maintain 150 ng/well and/or normalizelevels of CMV promoter-based plasmids across conditions. Candidateligands were added at 4000-fold dilution 8 h post-transfection.Luciferase activities were determined and normalized to theβ-galactosidase control. Data represent the mean±SD of triplicateassays.

Preparation of DAF-9 and Control Microsomes from Sf9 Cells

Sf9 (2×10⁶ cells/ml) were cultured in SF-900 SFM and were infected withbaculovirus at an MOI of 2-4. Cells were supplemented with 0.5 μg/mLhemin chloride, 100 μM δ-amino-levulinic acid, and 100 μM ferric citrateat the time of infection. Infected cells were harvested 60 hourspost-infection and microsomes prepared according to methodologydescribed, for instance, by Hood et al. (1996).

DAF-9 Microsomal Incubations

Microsomes containing hOR or hOR and Daf-9 generated from Sf9 cells werethawed on ice and added to a final concentration of 0.5 mg/mL in 0.1Mpotassium phosphate buffer in the presence of an NADPH regeneratingsystem (50 U/mL DL-isocitrate dehydrogenase, 0.1 M isocitrate and 0.1 MMgCl₂). Substrates were added from stock solutions at 10⁻² M to a finalconcentration of 100 μM (final reaction volume of 0.5 mL). The mixturewas preincubated for 3 min at 37° C. and the reaction was initiated bythe addition of NADPH (1 mM). After 16 hrs, the reactions were stoppedby extraction with 2×2 mL of methyl-tert-butyl-ether and the top layerswere combined and dried under nitrogen. In some experiments, 0.5 μg of1,4-cholestadiene-3-one was added as an internal standard for theextraction.

Rescue Assays

Microsomal reactions were extracted with methyl-tert-butyl ether, driedunder nitrogen, resuspended in 50 μl methanol, and mixed with 5×concentration of HB101 bacterial paste. The resulting mixtures werevacuum dried, resuspended in 100 μl of 5× concentrated HB101, and platedon 3 cm plates containing 4 mL NG agar. For rescue, ˜200 embryos from a4-8 hour egg laying were transferred onto the dried bacterial lawn.Mixtures of daf-9(+),gfp(+) and daf-9(−),gfp(−) embryos were placed onagar plates containing a mixture of bacteria and extracts from eitherDAF-9 or control microsomal reactions. daf-9(+),gfp(+) animals wereremoved after 48 hrs and the remaining daf-9(−),gfp(−) animals werelater scored for dauer arrest. For rescue experiments using puresteroids, 10 μl of compounds were mixed with 5× (90 μl) concentratedOP50 bacteria and plated. Final concentrations in the agar werecalculated as equally distributed over the total volume of agar (3-4mL/plate). Strains tested were grown reproductively onto regular NG agarfor 2 generations at 20° C.

C. elegans Lipid Extracts

Worms were grown on twenty 10 cm NGM plates seeded with HB101 bacteria.Gravid adults were bleached and the resulting embryos incubated in 2.8 LFernbach flasks containing 100-350 mL S-medium supplemented with 5 μg/mLNystatin, 50 μg/mL streptomycin sulfate overnight to allowsynchronization of LIs (Stiernagel, 1999). Two successive rounds ofgrowth (with 1-2% HB101) and lysis of gravid adults were performed until˜100-300 million synchronized L1 larvae were obtained. Final growth tothe L3/L4 stage was performed in a 15 L New Brunswick BiofloIVfermentor, with a working volume of 10.5 L at 20° C. with agitation (100RPM, 25% O2 saturation). Worms were harvested and bacteria and debriswere removed by sucrose flotation, then frozen in liquid nitrogen andstored at −80° C. Thawed worms were lyophilized for measurement of dryweight, resuspended in 0.1M NaCl and homogenized using an Emulsi-flexC-5 homogenizer (Avestin, Ottawa, Canada). Total lipids were extractedusing 2:1 chloroform:methanol. The resulting chloroform layer wascollected and back-extracted with two-thirds volume of water. The finalchloroform layer was dried with Na₂SO₄, filtered through Whatman filterpaper and then concentrated in vacuo. The resulting lipid extract wasre-suspended in chloroform and adsorbed to a silica column. Lipids wereeluted from the column in three fractions using 100 mL chloroform, 200mL 9:1 acetone:methanol, and 200 mL methanol. The 9:1 acetone:methanolextract was further fractionated by silica chromatography usingchloroform and increasing concentrations of methanol to 100%. Fractionswere dried under a stream of N₂ and tested for DAF-12 activation.

LC/MS Analysis

Samples were analyzed by LC/MS using a DAD in tandem with an MSD singlequadrupole instrument (Agilent Technologies, Palo Alto, Calif.) withAPI-ES in both positive and negative ion modes. Samples were dissolvedin methanol and loaded onto a pre-column (Zorbax C₈, 4.6×12.5 mm, 5 μm,Agilent) at 4 ml/min for 1 min with 30:70 methanol/water, bothcontaining 5 mM NH₄Ac, and then back flushed onto the analytical columnat 0.4 ml/min (Zorbax C₁₈, 4.6×50 mm, 5 μm, Agilent). The mobile phaseconsisted of methanol (A) and methanol/acetonitrile/water (60:20:20)(B), both containing 5 mM (NH₄Ac). The following gradient was run for atotal of 20 min: 0-6.5 min, 75% to 100% (A); 6.5-18 min, 100% (A);18.1-20 min, 75% (A). MS parameters were as follows: gas temperature350° C., nebulizer pressure 30 psig, drying gas (nitrogen) 12 L/min,VCap (positive and negative) 4000V, fragmentor voltage 150V (positiveions) or 200V (negative ions). For experiments in scan mode (positive ornegative), mass ranges between m/z 250-500 were used. Using selectiveion monitoring (in positive ion mode), signals for [M+H]⁺ ions wereobserved for 4-cholesten-3-one (m/z 385, retention time (RT) 12.5 min),lathosterone (m/z 384, RT 14.0 min), 1,4-cholestadien-3-one (m/z 383, RT10.2 min), (25R/S),26-hydroxy-4-cholesten-3-one (m/z 401, RT 5.7 min),(25R/S),26-3-keto-4-cholestenoic acid. Selective ion monitoring (SIM) innegative ion mode gave signals for [M−H]⁻ ions of(25R/S),26-3-keto-4-cholestenoic acid (m/z 413, RT 4.0 min). Bothpositive and negative ions were monitored simultaneously for samples runin SIM mode. Separation of 4-cholesten-3-one oxysterols was achieved asdescribed (Uomori et al., 1987). Briefly, samples were loaded onto thepre-column as described above and back flushed onto a TSK-gel ODS-120Tcolumn (4.6×250 mm, 5 μm, Tosoh Biosep, Montgomeryville, Pa.) running 1ml/min 7% water in methanol (both with 5 mM NH₄Ac). Total run time was30 min and the analytes were monitored by UV absorbance at 240 nm.

Binding Assays

DAF-12 ligand binding domain (aa 507-753) was expressed in BL21(DE3)cells as a 6×His-GST fusion protein using pET24a (Novagen). Ligandbinding was determined by AlphaScreen assays from Perkin-Elmer (Xu etal., 2002) with approximately 40 nM receptor and 40 nM of biotinylatedSRC1-4 (QKPTSGPQTPQAQQKSLLQQLLTE) peptide in the presence of 5 μg/mLdonor and acceptor beads in a buffer containing 50 mM MOPS, 50 mM NaF,50 mM CHAPS, and 0.1 mg/mL bovine serum albumin at pH 7.4. EC₅₀ bindingvalues were determined from nonlinear least square fit of the data basedon an average of three experiments.

To screen for sterol-derived ligands of DAF-12 a co-transfection assayin HEK293 cells was performed using a chimeric GAL4-DAF-12 receptor anda GAL4-responsive luciferase reporter. Assays were performed in thepresence or absence of co-transfected DAF-9 due to the strong geneticevidence suggesting it plays an important role in the synthesis of theDAF-12 ligand (Gerisch and Antebi, 2004; Gerisch et al., 2001; Gill etal., 2004; Jia et al., 2002; Mak and Ruvkun, 2004). Our initial compoundscreen consisted of bile acids and known vertebrate steroid andendocrine hormones as these compounds are ligands for PXR, VDR and LXR,the closest vertebrate homologs of DAF-12 (Antebi et al., 2000;Mooijaart et al., 2005).

The initial screen identified the bile acid, 3-keto-lithocholic acid(3K-LCA, FIG. 1A) as a weak, micromolar activator of DAF-12 independentof DAF-9 (FIG. 1B). Interestingly, lithocholic acid (LCA), which differsfrom 3K-LCA by an α-hydroxyl group at C-3, did not exhibit activity onits own or in the presence of co-transfected DAF-9 (FIG. 1B). Theseresults suggested that a C-3 ketone was required for DAF-12 activationby 3K-LCA. No other bile acids or endocrine hormones tested activatedDAF-12, including cholic acid (CA), chenodeoxycholic acid (CDCA),deoxycholic acid (DCA), 6-keto-lithocholic acid (6K-LCA),7-keto-lithocholic acid (7K-LCA), progesterone, pregnenolone,testosterone, estradiol, corticosterone, 1,25-dihydroxyvitamin D3, and20-hydroxyecdysone (FIG. 1B).

The foregoing data suggested that endogenous 3-keto sterols from C.elegans are candidate DAF-12 ligands. Lathosterol and its4-methyl-derivative, lophenol, are cholesterol metabolites that havedistinct effects on the nematode life cycle (Chitwood et al., 1983;Matyash et al., 2004; Merris et al., 2003). When given as the soledietary sterol, lathosterol supported full reproductive growth (Merriset al., 2003), while worms grown only in the presence of lophenolconstitutively entered dauer diapause (Matyash et al., 2004). Thesestudies suggest lathosterol but not lophenol may be a direct precursorto the DAF-12 ligand. Since activation by 3K-LCA required a C-3 ketone,lathosterol and lophenol were tested along with their respective 3-ketoderivatives, lathosterone and lophenone, for activity in theco-transfection assay. Upon co-transfection with DAF-9, activation ofDAF-12 was markedly increased by lathosterone (433-fold) and lophenone(103-fold), but not by their respective 3β-hydroxy derivatives (FIG.1C). In addition, 4-cholesten-3-one, a natural oxidation product ofcholesterol, activated DAF-12 (109-fold) in the presence of DAF-9 (FIG.1C). Unlike 3K-LCA, activation of DAF-12 by lathosterone, lophenone, and4-cholesten-3-one required the presence of DAF-9. Structurally, 3K-LCAdiffers from these 3-keto-sterols in the length and oxidation state ofthe side chain and in the saturation of the sterol nucleus (FIG. 1A).These results revealed that DAF-9 metabolizes 3-keto-sterols, possiblythrough side-chain oxidation, into DAF-12 activators.

DAF-9 Metabolites of 3-Keto-sterols Rescue daf-9 Null Worms

The 3-keto-sterol metabolites were tested for their ability to rescuethe Daf-c and Mig phenotypes of daf-9 null animals (Albert and Riddle,1988; Gerisch et al., 2001; Jia et al., 2002). Individual 3-keto sterolsand their respective 3β-hydroxy sterols were incubated with Sf9 cellmicrosomes co-expressing DAF-9 and the human P450 oxidoreductase (hOR).As a control, microsomes from cells expressing hOR alone were used. Forrescue experiments daf-9(dh6)dhEx24, which carries an unstableextrachromosomal array of daf-9(+) and the nuclear marker sur-5:GFP, wasutilized (Gerisch et al., 2001). Extracts from DAF-9 microsomesincubated with either 4-cholesten-3-one or lathosterone resulted in 100%rescue of the Daf-c phenotypes of animals lacking the daf-9(+) array(FIG. 1D). Remarkably, these animals were indistinguishable fromwild-type adults. Indeed, they bypassed dauer, exhibited normal gonadalmigration, and produced all Daf-c progeny upon passage to plates lackingmicrosomal extracts. Rescue by 4-cholesten-3-one and lathosteronerequired metabolism by DAF-9, since incubation of these sterols withcontrol microsomes did not rescue any daf-9 phenotypes. Interestingly,extracts from DAF-9 microsomes incubated with lathosterol only partiallyrescued (83%) the Daf-c phenotype (FIG. 1D). However, the rescuedanimals exhibited the Mig phenotype and were sterile. This effect alsorequired DAF-9 as no effect was seen after incubation of lathosterolwith control microsomes. Finally, although incubation of lophenone withDAF-9 microsomes resulted in rescue of Daf-c, none of these animals werenormal (58% were Mig and the remaining 42% failed to enter dauer, hadmolting defects, or were dead) (FIG. 1D). This effect was dependent onDAF-9 and the C-3 ketone in lophenone, since lophenol had no effect ondaf-9 animals after incubation with DAF-9 or control microsomes.Notably, no effect was seen after incubation of cholesterol with DAF-9or control microsomes. Altogether, the in vivo rescue assays revealedthat DAF-9 microsomes metabolize 4-cholesten-3-one and lathosterone intoactivities that completely rescued both the Daf-c and Mig phenotypes ofdaf-9 null animals.

Identification of DAF-9 Metabolites of 4-Cholesten-3-one

The data above suggested that DAF-9 has enzymatic activity thatmetabolizes 4-cholesten-3-one and lathosterone into DAF-12 ligands.Although the absolute potency of these DAF-9 metabolites could not bedetermined using the rescue assay, dose response curves from theco-transfection assay revealed that lathosterone metabolites were eithersignificantly more potent or more abundantly produced than4-cholesten-3-one metabolites (FIG. 1E). DAF-9 metabolites wereidentified with liquid chromatography/mass spectrometry (LC/MS). The3-keto-Δ⁴-enone structure present in 4-cholesten-3-one has significantUV absorbance at 240 nm, permitting detection of the metabolites.Incubation of 4-cholesten-3-one with DAF-9 microsomes yielded two newpeaks at 240 nm that were not present in the control microsomes (FIG.2A). These metabolites eluted at 4.0 (peak 1) and 5.5 (peak 2) minuteson a reverse phase C₁₈ column, indicating they were oxygenatedderivatives of 4-cholesten-3-one which elutes at 13 min (FIG. 2A). Sincelathosterone is not UV active, its DAF-9 metabolites were scanned innegative ion mode revealing two peaks that were not present in thecontrol microsome reactions. DAF-9 metabolites of lathosterone elutedmuch earlier than lathosterone at 4.0 and 4.2 minutes (peaks 3 and 4),analogous to the pattern seen for 4-cholesten-3-one (FIG. 2B). Fractionsfrom DAF-9 microsomal reactions subjected to reverse phase-HPLC werealso tested for DAF-12 activation and rescue of daf-9 null animals (FIG.2C-F). Fractions corresponding to peaks 1-4 (i.e., the 4-cholesten-3-oneand lathosterone metabolites) activated DAF-12 several hundred-foldindependent of DAF-9 (FIGS. 2C and D) and rescued daf-9 null animals(FIGS. 2E and F). Interestingly, the lathosterone metabolites werestronger at activating DAF-12 and rescuing daf-9, suggesting thesemetabolites may be more potent.

Next, LC/MS was used as a first step in the identification of theactivities in peaks 1-4. Based on the molecular weight of4-cholesten-3-one ([M+H]⁺ m/z=385) and the retention times and massspectra of the new metabolites, peak 2 is consistent with amono-hydroxylated derivative of 4-cholesten-3-one ([M+H]⁺ m/z=401) andpeak 1 is consistent with a carboxylic acid derivative ([M+H]⁺ m/z=415)(FIG. 2G). Further evidence that peak 1 represented a carboxylic acidmetabolite was found after a negative ion scan in which a unique peak at4.0 min was found only in the DAF-9 microsomes with a base peak at m/z413 (FIG. 2G). Peak 4, which was scanned in negative ion mode, yieldedsimilar mass spectra to peak 1, consistent with the conclusion that peak4 is the carboxylic acid derivative of lathosterone (FIG. 2H). Finally,peak 3 contained one DAF-9 specific metabolite at m/z 415 (FIG. 2H).Although the identity of this peak remains unknown, the observedactivity tracks predominantly with peak 4 and not peak 3.

DAF-9 is a 3-Keto-sterol-26-monooxygenase

The finding that 3K-LCA was a weak activator of DAF-12 suggested thatthe position of oxidation of DAF-9 metabolites was on the side-chain.The commercial availability of monohydroxylated derivatives ofcholesterol permitted us to focus on defining the site of oxidation onthe 4-cholesten-3-one metabolites of DAF-9. A panel of side-chainoxidized 4-cholesten-3-one derivatives was generated by converting5-cholesten-3β-ol (Ring A, 3β-hydroxy-Δ⁵) oxysterols (20(S)—OH—, 22(R)—OH—, 22(S)—OH—, 24-OH—, 25-OH—, (25R),26-OH—, and(25S),26-OH-cholesterol) into their respective 4-cholesten-3-one (RingB, 3-keto-Δ⁴) oxysterols using cholesterol oxidase (FIG. 3A). Generationof 4-cholesten-3-one oxysterols was confirmed by MS ([M+H]⁺ m/z 401) andUV spectra (240 nm). When tested in the co-transfection assayindependent of DAF-9, two diastereomers of 26-hydroxy-4-cholesten-3-one((25S),26-hydroxy-4-cholesten-3-one and(25R),26-hydroxy-4-cholesten-3-one) were strongly active (FIG. 3B). Incontrast, the corresponding Ring A oxysterols were inactive, confirmingthe idea that DAF-12 ligands require a 3-keto group. Chromatographicseparation of the Ring B oxysterol panel and comparison to the4-cholesten-3-one metabolites of DAF-9 resolved peak 2 into two peaksthat co-eluted with the diastereomers of 26-hydroxy-4-cholesten-3-one(FIG. 3C). These results revealed that DAF-9 is a non-stereoselective4-cholesten-3-one 26-hydroxylase.

Given that DAF-9 microsomes produced both hydroxylated and carboxylatedmetabolites of 4-cholesten-3-one, the possibility that DAF-9 oxidizes4-cholesten-3-one at C-26 to produce both diastereomers of26-hydroxy-4-cholesten-3-one and then subsequently oxidizes them againinto carboxylic acids was investigated. Indeed, incubation of either(25S) or (25R),26-hydroxy-4-cholesten-3-one with DAF-9 microsomesresulted in the production of a single major UV active peak with 4 minretention times and mass spectra at m/z 415 in positive ion mode (FIG.3D). These retention times and mass spectral properties were identicalto the carboxylated metabolite found after incubation 4-cholesten-3-onewith DAF-9 microsomes (FIG. 2A, peak 1) and were not detected in controlmicrosomal reactions (FIG. 3D). Conversion of the diastereomers of26-hydroxy-4-cholesten-3-one into their C-26 carboxylic acids required a3-keto-Δ⁴ structure as the 3β-hydroxy-Δ⁵-sterols,(25R),26-hydroxycholesterol and (25S),26-hydroxycholesterol, were notoxidized into their carboxylated metabolites. Finally, incubation ofeither diastereomer of 26-hydroxy-4-cholesten-3-one with DAF-9dramatically increased their ability to rescue daf-9 phenotypes.Extracts from control microsome incubations with(25R),26-hydroxy-4-cholesten-3-one had no biological effect, while(25S),26-hydroxy-4-cholesten-3-one produced only an incomplete rescue(10% molt defects, 75% sterile Mig adults), resembling the activity inpeak 2 of FIG. 2A (FIG. 3E). In contrast, incubation of either sterolwith DAF-9 microsomes resulted in complete rescue of Daf-c and Migphenotypes in 100% of animals tested (FIG. 3E). Taken together, theseresults demonstrated that DAF-9 metabolizes 4-cholesten-3-one into daf-9rescuing activities through successive oxygenations at C-26, resultingin the production of carboxylic acid metabolites.

DAF-9 and Mammalian CYP27A1 are Functional Orthologs

Successive oxidation of sterol side chains has been demonstrated for themammalian cytochrome P450, CYP27A1. Like DAF-9, CYP27A1 successivelyoxidizes sterol substrates at C-26 to produce both a (25R),26-hydroxymetabolite (e.g., 27-hydroxycholesterol) and (25R),26-carboxylic acidmetabolite (e.g., cholestenoic acid) (Cali and Russell, 1991). Notably,in vitro studies have shown that CYP27A1 utilizes 4-cholesten-3-one moreefficiently than cholesterol (Norlin et al., 2003). Therefore, theability of CYP27A1 to metabolize 4-cholesten-3-one into a DAF-12activator was tested. Co-transfection of HEK293 cells with GAL4-DAF-12,human or mouse CYP27A1, and bovine adrenodoxin resulted in potentactivation of the GAL4-DAF-12 (FIG. 8). In contrast, DAF-12 was notactivated by 4-cholesten-3-one in the presence of bovine adrenodoxinalone. Interestingly, in the presence of 25 μM lathosterone,co-transfection of CYP27A1 had no effect. These results revealed thatDAF-9 and CYP27A1 have similar enzymatic activities and overlapping, butdistinct, substrate specificities.

3-Keto-4-cholestenoic Acid is a Hormonal Activator of DAF-12

To confirm the identity of the most potent DAF-9 metabolites as C-26carboxylic acids of 4-cholesten-3-one (i.e., 3-keto-4-cholestenoicacid), the diastereomers of 3-keto-4-cholestenoic acid (FIG. 4A) wassynthesized and tested along with the diastereomers of26-hydroxy-4-cholesten-3-one, to transactivate DAF-12. The syntheticcompounds exhibited chromatographic and mass spectral propertiesidentical to the acidic metabolites obtained from incubation of4-cholesten-3-one with DAF-9 microsomes (FIG. 9). When tested in theco-transfection assay, DAF-12 responded to all 4 steroids with thefollowing rank order of potencies: (25S),26-3-keto-4-cholestenoic acid(EC50=100 nM); (25R),26-3-keto-4-cholestenoic acid (EC50=1 μM);(25S),26-hydroxy-4-cholesten-3-one (EC50=1 μM);(25R),26-hydroxy-4-cholesten-3-one (EC50=2 μM) (FIG. 4B). Activation by(25S),26-3-keto-4-cholestenoic acid was specific to DAF-12 and notobserved with other nuclear receptors tested, including the closest C.elegans, Drosophila, and human homologs (FIG. 10). In addition, theDAF-12 ligand binding domain mutants (R564C, R564H) were dramaticallyattenuated in their response to (25S),26-3-keto-4-cholestenoic acid(FIG. 10) and did not respond to (25R),26-3-keto-4-cholestenoic acid.

Consistent with its function as a DAF-12 hormonal ligand,(25S),26-3-keto-4-cholestenoic acid rescued Daf-c and Mig daf-9phenotypes (FIGS. 4C and D). At hormone concentrations of 250 nM, daf-9animals were indistinguishable from wild type: they bypassed dauerdiapause to become reproductive adults (FIGS. 4C and D), and producedbroods comparable to wild type (˜300 offspring, n=5 worms). Rescuedanimals also exhibited properly turned gonads, lacked dauer alae, anddisplayed normal pharyngeal expansion (FIG. 4C). When placed on platesseeded without hormone, animals produced all dauer progeny, confirmingtheir genotype as daf-9 null and demonstrating a lack of maternalrescue. Similarly, the Mig phenotype of the weak allele daf-9(rh50) wasreversed at 250 nM hormone (FIG. 4E). At intermediate concentrations(50-100 nM), a proportion of null mutants exhibited Mig and moltingdefects, suggesting these phenotypes arise from a reduction in hormonelevels (FIG. 4D). The 25R diastereomer of 3-keto-4-cholestenoic acidalso rescued daf-9 phenotypes, albeit at roughly 5-10-fold higherconcentrations (FIG. 4D). Consistent with the data above (FIG. 3E),(25R),26-hydroxy-4-cholesten-3-one had no effect on daf-9 nulls, even atconcentrations as high as 33 μM, while(25S),26-hydroxy-4-cholesten-3-one caused 93% of worms (n>60) to bypassdauer, but still exhibit Mig and/or molting defects, and sterility.These results revealed that 3-keto-4-cholestenoic acid functions as a C.elegans hormone that inhibits dauer formation and promotes reproductivedevelopment.

As predicted by co-transfection assays (FIG. 10), daf-12 LBD mutantswere compromised in their ability to respond to(25S),26-3-keto-4-cholestenoic acid (FIG. 4E). For example, hormone hadno effect on daf-12(rh61), which truncates the receptor ligand bindingdomain and results in strong Mig phenotypes (FIG. 4E). Another mutant,daf-12(rh273) contains a missense lesion in a predicted ligand contactsite, and gives Daf-c, Mig, and molting defects. Interestingly, at >250nM (25S),26-3-keto-4-cholestenoic acid, Daf-c but not Mig or moltphenotypes were rescued (FIGS. 4E and F), consistent with the conclusionthat this mutation decreases ligand binding affinity.

3-Keto-4-cholestenoic Acid Acts Downstream of Insulin, TGFβ, andCholesterol Lysosomal Transport Pathways

To investigate further the biological activity of 3-keto-4-cholestenoicacid the ability of (25S),26-3-keto-4-cholestenoic acid to rescue thedauer constitutive phenotypes of worms carrying mutations in theinsulin-like receptor and TGFβ signaling genes positioned upstream ofdaf-9 and daf-12 was tested. Accordingly, it was found that(25S),26-3-keto-4-cholestenoic acid rescued the Daf-c phenotypes ofdaf-7(m62)/TGFβ peptide and daf-2/insulin-like receptor mutants (FIG.4F). Whereas weak daf-2(e1368) mutants responded similar to daf-9 bypassing dauer diapause and becoming gravid adults, the strongdaf-2(e1370) mutants circumvented dauer morphogenesis but remaineddevelopmentally arrested as dark L3-like larvae, even at the highestconcentrations. These results imply that insulin/IGF signaling mustimpinge upon the pathway both upstream, downstream, or parallel tohormone production. Finally, (25S),26-3-keto-4-cholestenoic acideffectively rescued the Daf-c phenotypes of ncr-1;ncr-2 double mutants(FIG. 4F), which encode homologs of the human Niemann-Pick type C1lysosomal transport protein involved in sterol transport (Li et al.,2004).

3-Keto-4-cholestenoic Acid Binds DAF-12 as a Bona Fide Ligand

A hallmark of nuclear receptor agonists is their ability todiametrically regulate interactions with co-repressors andco-activators. DIN-1S, a homolog of the mammalian co-repressor, SHARP,is thought to function as a DAF-12 co-repressor in the absence of ligandand thereby repress target genes that promote reproduction (Ludewig etal., 2004). To test this hypothesis, a mammalian two-hybrid assay wasdesigned. In the absence of ligand, GAL4-DIN-1S interacted withVP16-DAF-12 as predicted (FIG. 5A). This interaction was completelyabolished by addition of 1 μM (25S),26-3-keto-4-cholestenoic acid,supporting the conclusion that the hormone disrupts dauer-promotingcomplexes involving DAF-12 and DIN-1S. Importantly, hormone did notdisrupt the interaction between GAL4-DIN-1S and a VP16-DAF-12 proteincarrying the R564C mutation (FIG. 5A). Likewise, it was observed thatDIN-1S-dependent repression of GAL4-DAF-12 basal activity could bereversed by addition of 100 nM (25S),26-3-keto-4-cholestenoic acid (FIG.5B). A similar two-hybrid analysis was used to show hormone-dependentrecruitment of co-activator to DAF-12. (25S),26-3-Keto-4-cholestenoicacid caused a ligand-dependent interaction between the fourth receptorinteraction domain (ID4) of the mammalian co-activator protein SRC-1(GAL4-SRC-1 ID4) and VP16-DAF-12, but not VP16-DAF-12-R564C (FIG. 5C).Also tested was the ability of the hormone to transactivate full-lengthDAF-12 on a luciferase reporter plasmid containing the DAF-12 bindingsites of Lit-1 kinase, a proposed DAF-12 target gene (Shostak et al.,2004). In the presence of (25S),26-3-keto-4-cholestenoic acid, DAF-12transactivated the Lit-1 kinase reporter plasmid in a dose-dependentmanner (EC50=100 nM) (FIG. 5D).

Finally, to determine the in vitro ligand binding properties of DAF-12with the various 4-cholesten-3-one metabolites shown in FIG. 4A, anAlpha Screen assay that can detect ligand-dependent interactions betweenreceptors and co-activator peptides was employed (Xu et al., 2002). At 1μM, the (25S) and (25R),26-3-keto-4-cholestenoic acids produced 58-foldand 24-fold increases in binding units, respectively, compared tovehicle control (FIG. 6A). In contrast, the hormone precursor,4-cholesten-3-one, and the hydroxyl intermediate,(25S),26-hydroxy-4-cholesten-3-one, showed no significant binding. Weakbinding activity (2-fold) was detected for the weaker activatingdiastereomer, (25R),26-hydroxy-4-cholesten-3-one. Saturation bindingkinetics revealed that (25S),26-3-keto-4-cholestenoic acid binds DAF-12with high affinity (EC50=1 nM) (FIG. 6B). A similar analysis revealed(25R),26-3-keto-4-cholestenoic acid binds DAF-12, but with at least a10-fold lower affinity. Although the C-26 carboxylic acid derivative oflathosterone is not available, a strong ligand binding activity wasdetected in the DAF-9 microsomes that were incubated with lathosterone(FIG. 6C) and were shown to contain the presumed carboxylic acidmetabolite (FIG. 2). These results support the conclusion that both the3-keto-4-cholestenoic acid and lathosterone carboxylic acid hormonesmediate their effects in vivo through direct binding to DAF-12.

3-Keto-4-cholestenoic Acid is an Endogenous C. elegans Hormone

To determine whether the DAF-12 ligands identified above are present invivo, lipid extracts from L3-L4 staged worms were generated and testedfor DAF-12 activity in a co-transfection assay. Crude extractscontaining activity were fractionated by silica column chromatography(FIG. 7A) and the DAF-12 activating fraction was determined to be in theacetone:methanol eluate (FIG. 7B). Subsequent fractionation of thisactivity revealed the presence of two distinct DAF-12 activity peaks atfractions 30-33 and 57-63 (FIG. 7C). Although the level of activity infraction 30-33 was too low for further analysis, its chromatographicproperties were consistent with the alcohol derivatives of4-cholesten-3-one and lathosterone. After pooling and re-purification byHPLC of fractions 57-63, however, enough material was obtained to lookfor the presence of the carboxylic acid derivatives by LC/MS (FIG. 7D,upper panel). Selective ion monitoring (SIM) identified a peak at m/z413 in negative ion mode with a retention time similar to the3-keto-4-cholestenoic acid metabolite of DAF-9 (FIG. 7D, middle panel).This signal correlated with DAF-12 activity, as it was not present inneighboring fractions that lacked activity. In addition, a second,higher abundance peak was detected that co-migrated with the carboxylicacid of lathosterone (FIG. 7D, bottom panel). As expected, lipids fromthese pooled fractions rescued the Daf-c and Mig phenotypes in 100% ofdaf-9 null worms tested (n>300).

Transactivation of C. elegans nuclear hormone receptor DAF-12 by(25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic S acid),3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic R acid), and3β-ol-5-cholesten-25(S)-26-oic acid.

HEK cells were transfected in 96 well plates with plasmids as follows:50 ng of mh100-tk-luc reporter, 10 ng CMX-β-galactosidase reporter, 15ng CMX-Gal-4-c.eDAF-12LBD which expresses a fusion protein of Gal4DNA-binding domain with ligand-binding domain of C. elegans nuclearhormone receptor DAF-12. Indicated dose of ligands were added 6 hourslater and luciferase as well as β-galactosidase activities were measured24 hours after transfection. Relative luciferase unit (RLU) was thencalculated by normalizing the luciferase activity to the β-galactosidasecontrol. The transactivation of the nuclear hormone receptor wasrepresented by fold change of RLU induced by treatment of indicatedconcentration of ligands. Data represent the mean±S.D. of triplicateassays (FIG. 12).

Infections by hookworms, which are parasitic nematodes, are a majorreason for anemia and growth retardation, especially in children andpregnant women. Hookworms and C. elegans belong to the same evolutionaryclade and have many features in common. In particular, the infective L3(iL3) larval stage, the only one during hookworm development when theparasite is able to infect a host, is similar to the dauer larval stageof C. elegans.

The present inventors have cloned DAF-12 homologs from three notorioushookworm species, Necator americanus, Ancylostomiasis ceyelanicum, andA. caninum. These homologs were evaluated to assess their susceptibilityto activation by dafachronic acids, as well as by a structurally-relatedsteroid analog, called “3β-5-cholestenoic acid,” which is found inhookworm hosts (i.e., humans, hamsters, and dog).

As illuminated in the following examples, the data show that thesecompounds are able to activate all three hookworm homologs of DAF-12,thereby evidencing a conserved signaling pathway that controls theinfectious and adult stages in hookworm as it does in C. elegans. Thus,the data underscore the targeting of these homologs as a feasiblestrategy for pharmacological intervention of hookworm infection, whichaffects over one billion people worldwide.

Transactivation of human hookworm N. americanus nuclear hormone receptorDAF-12 by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic S acid),3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic R acid), and30-ol-5-cholesten-25(S)-26-oic acid.

HEK cells were transfected in 96 well plates with plasmids as follows:50 ng of mh100-tk-luc reporter, long CMX-β-galactosidase reporter, 15 ngCMX-Gal4-n.a.DAF-12LBD which expresses a fusion protein of Gal4DNA-binding domain with ligand-binding domain of N. americanus nuclearhormone receptor DAF-12. Indicated dose of ligands were added 6 hourslater and luciferase as well as β-galactosidase activities were measured24 hours after transfection. Relative luciferase unit (RLU) was thencalculated by normalizing the luciferase activity to the β-galactosidasecontrol. The transactivation of the nuclear hormone receptor wasrepresented by fold change of RLU induced by treatment of indicatedconcentration of ligands. Data represent the mean±S.D. of triplicateassays (FIG. 13).

Transactivation of human/hamster hookworm A. ceyelanicum nuclear hormonereceptor DAF-12 by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic Sacid), 3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic Racid), and 3β-ol-5-cholesten-25(S)-26-oic acid.

HEK cells were transfected in 96 well plates with plasmids as follows:50 ng of mh100-tk-luc reporter, 10 ng CMX-β-galactosidase reporter, 15ng CMX-Gal-4-a.ceyDAF-12LBD which expresses a fusion protein of Gal4DNA-binding domain with ligand-binding domain of A. ceyelanicum nuclearhormone receptor DAF-12. Indicated dose of ligands were added 6 hourslater and luciferase as well as β-galactosidase activities were measured24 hours after transfection. Relative luciferase unit (RLU) was thencalculated by normalizing the luciferase activity to the β-galactosidasecontrol. The transactivation of the nuclear hormone receptor wasrepresented by fold change of RLU induced by treatment of indicatedconcentration of ligands. Data represent the mean±S.D. of triplicateassays (FIG. 14).

Transactivation of dog hookworm A. caninum nuclear hormone receptorDAF-12 by (25S),26-3-keto-4-cholestenoic acid (Δ⁴ dafachronic S acid),3-keto-7,(5a)-cholesten-25(R)-26-oic acid (Δ⁷ dafachronic R acid), and3β-ol-5-cholesten-25(S)-26-oic acid.

HEK cells were transfected in 96 well plates with plasmids as follows:50 ng of mh100-tk-luc reporter, 10 ng CMX-β-galactosidase reporter, 15ng CMX-Gal4-a.c.DAF-12LBD which expresses a fusion protein of Gal4DNA-binding domain with ligand-binding domain of A. caninum nuclearhormone receptor DAF-12. Indicated dose of ligands were added 6 hourslater and luciferase as well as β-galactosidase activities were measured24 hours after transfection. Relative luciferase unit (RLU) was thencalculated by normalizing the luciferase activity to the β-galactosidasecontrol. The transactivation of the nuclear hormone receptor wasrepresented by fold change of RLU induced by treatment of indicatedconcentration of ligands. Data represent the mean±S.D. of triplicateassays (FIG. 15).

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1. A compound of formula:

or a stereoisomer or pharmaceutically acceptable salt thereof.
 2. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound selected from the group consisting of:

stereoisomers, and pharmaceutically acceptable salts thereof.